Methods and compositions for treating neurological disease

ABSTRACT

This invention relates to methods and compositions for treating neurological disease, and more particularly to methods of delivering iRNA agents to neural cells for the treatment of neurological diseases.

RELATED APPLICATIONS

This application is continuation of co-pending U.S. Utility applicationSer. No. 11/506,448, entitled “Methods and Compositions for TreatingNeurological Disease,” filed Aug. 18, 2006, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 60/709,985, entitled“Methods and Compositions for Treating Neurological Disease,” filed Aug.18, 2005 and U.S. provisional patent application Ser. No. 60/833,234,entitled “Methods and Compositions for Treating Neurological Disease,”filed Jul. 25, 2006. The entire contents of the above-referenced patentapplications are hereby incorporated by this reference.

GOVERNMENT SUPPORT

The work described herein was carried out, at least in part, using fundsfrom the U.S. government under grant number 38194 awarded by theNational Institutes of Health. The government may therefore have certainrights in the invention.

TECHNICAL FIELD

This invention relates to methods and compositions for treatingneurological disease, and more particularly to methods of deliveringiRNA agents to neural cells for the treatment of neurological diseases.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.,Nature 391:806-811, 1998). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function.

SUMMARY

Aspects of the invention relate to compositions for treating aneurological disorder, and methods of using those compositions. In oneaspect, the invention features a method of treating a subject having, orat risk for developing a neurological disorder by administering an iRNAagent that inhibits expression of a gene expressed in neural cells. Inone embodiment, the iRNA agent includes a conjugate to facilitate uptakeof the iRNA agent into neural cells. In a preferred embodiment, theconjugate is a lipophilic moiety, e.g., cholesterol. In anotherembodiment, the iRNA agent inhibits expression of the gene expressed ina neural cell that is involved in a neurological disease or disorder. Inyet another embodiment, the iRNA agent is used to treat a patient havingor at risk for developing a neurological disorder. In one embodiment,the iRNA agent modified for enhanced uptake into neural cells caninhibit, or decrease, expression of the huntingtin (htt) gene in a humanhaving or at risk for developing Huntington's Disease (HD).

In a preferred embodiment, the subject is a mammal, such as a human,e.g., a subject diagnosed as having, or at risk for developing, aneurological disorder.

In one embodiment the sense strand of the iRNA agent can include atleast one mismatch within the antisense strand of the oligonucleotideagent. The mismatch can confer an advantage on the iRNA agent, such asby enhancing antisense strand selection by the RNAi Induced SilencingComplex (RISC). In one embodiment, the mismatch is at least 1, 2, 3, 4,or 5 nucleotides away from the 3′-terminal nucleotide of the sensestrand.

In one embodiment, the iRNA agent includes an antisense strand that issubstantially complementary to a sequence encoded by a region of thehuman htt gene including or overlapping a sequence provided in GenBankAccession Number NM_(—)002111 (Aug. 8, 2005).

In certain embodiments, the iRNA agents can target an htt RNA and caninclude a sense and/or antisense sequence listed in Table 1 or Table 2.In preferred embodiments, the iRNA agent includes at least onemodification in addition to the lipophilic moiety for enhanced uptakeinto neural cells. The at least one additional modification can be,e.g., a phosphorothioate or 2′O-methyl (2′OMe) modification.

TABLE 1 iRNA Agents targeting htt SEQ IRNA  ID Agent ID Sequence^(a)Strand^(b)  NO: E1-4   5′-CCCUGGAAAAGCUGAUGACGG-chol S 13′-CUGGGACCUUUUCGACUACUU as 2 E1-4-b   5′-CCCUGGAAAAGCUGAUGACG S 33′-CUGGGACCUUUUCGACUACUU as 4 7246   5′-CCCUCAUCCACUGUGUGCCCU-chol S 53′-GAGGGAGUAGGUGACACACGU as 6 7246-b   5′-CCCUCAUCCACUGUGUGCCCU S 73′-GAGGGAGUAGGUGACACACGU as 8 T2886C-6   5′-UGUGCUGACUCUGAGGAAAAG-chol S9 3′-CUACACGACUGAGACUCCUUG as 10 T2886C-   5′-UGUGCUGACUCUGAGGAAAAG S 116-b 3′-CUACACGACUGAGACUCCUUG as 12 E1-3   5′-CCCUGGAAAAGCUGAUGAAGG-cholS 13 3′-CUGGGACCUUUUCGACUACUU as 14 E1-3-b    5′-CCCUGGAAAAGCUGAUGAAGG S15 3′-CUGGGACCUUUUCGACUACUU as 16 ^(a) ^(“)Chol” indicates cholesterolligand; underlined nucleotides are mismatched with respect to theantisense strand; bold nucleotides represent SNP locations ^(b) ^(“)s”indicates sense strand; “as” indicates antisense strand ^(a) ^(“)Chol”indicates cholesterol ligand; underlined nucleotides are mismatched withrespect to the antisense strand; bold nucleotides represent SNPlocations ^(b) ^(“)s” indicates sense strand; “as” indicates antisensestrand

TABLE 2 siRNAs targeting Huntington Oligonucleotide ligand conjugates

Ligand building blocks

Project/ SEQ ID ALN Mass Purity Target  NO: Seq. # Sequence 5′-3′ CalcFound CGE (%) htt 3005 5′ CsCCUGGAAAAGCUGAUGACsGsG 3′ 6822.3 6821.8 82.23006 5′  UsUCAUCAGCUUUUCCAGGGsUsC 3′ 6635.08 6634.63 85.6 htt 3007 5′ CsCsCUGGAAAAGCUGAUGAsCsGsG 3′ 6854.43 6853.89 81.1 3008 5′ UsUsCAUCAGCUUUUCCAGGsGsUsC 3′ 6667.21 6666.59 86.4 htt 3009 5′ UsGUGCUGACUCUGAGGAAAsAsG 3′ 6824.27 6821.95 90.7 3010 5′ GsUUCCUCAGAGUCAGCACAsUsC 3′ 6680.17 6979.8 88.2 htt 3011 5′ UsGsUGCUGACUCUGAGGAAsAsAsG 3′ 6856.40 6856.13 90.2 3012 5′ GsUsUCCUCAGAGUCAGCACsAUC 3′ 6712.30 6712.05 89.3 htt 3072 5′ CsCCU_(2′-OMe)GGAAAAGCU_(2′-oMe)GAU_(2′-OMe)GACsGsG 3′ 6864.38 6863.7987.9 3073 5′  UTOMeSGU_(2′-OMe)GCU_(2′-OMe)GACUCU_(2′-OMe)GAGGAA 6880.386879.82 85.7  AsAsG 3′ htt 3076 5′  CCCUCAUCCACUGUGUGCCCU 3′ 6520.86520.69 88.5 3077 5′  UGCACACAGUGGAUGAGGGAG 3′ 6854.15 6853.92 86.3 htt3108 5′ -CCACAUGAAGCAGCACGACUU-3 ′ 6678.06 6677.93 89.1 3109 5′ AAGUCGUGCUGCUUCAUGUGG_(2′-OMe)U_(2′-OMe) C  7345.37 7344.25 86.2 htt3075 5′  CsCCUGGAAAAGCUGAUGACsGsGs-Chol 3′ 7542.37 7543.06 98.7 3112 5′ Cy-3sCCCUGGAAAAGCUGAUGACsGsGs-Chol 3′ 8363.08 8363 & 93.78 8179 htt3137 5′  CsCCUGGAAAAGCUGAUGACGsGs-Chol 3′ 7526.30 7526.92 84.2 3142 5′ CsCCUCAUCCACUGUGUGCCCsUs-Chol 3′ 7273.06 7273.91 82.0 htt 3144 5′ Cy-3 sCsCCUCAUCCACUGUGUGCCCsUs-Chol 3′ 7909.8 7961.3 89.0 3110 5′ CCACAUGAAGCAGCACGACUU-Chol 3′ 7382.06 7382.91 84.73 htt 3074 5′ Cy-3-sCCCUGGAAAAGCUGAUGACs Gs G 3′ 7459.08 7458.07 78.43 3111 5′ Cy-3-AAGUCGUGCUGCUUCAUGUGG_(2′-OMe)U_(2′-OMe)C 3′ 7981.37 7982.74 82.0htt 3112 5′  Cy-3-sCCC UGG AAA AGC UGA UGA CsGsGs Chol 3′ 8363.08 8363 &93.78 8179 3139 5′  Cy-3 sCCCUGGAAAAGCUGAUGACGsGsChol 3′ 8163.1 8165.089.2 htt 3143 5′  UsGCACACAGUGGAUGAGGGAsGs-Cy-3 3′ 7576.4 7577.0 81.23138 5′  UsUCAUCAGCUUUUCCAGGGUsCs-Cy-3 3′ 7309.1 7308.3 86.0 “s”indicates phosphorothioate; ′N₂ _(,-) _(OMe) _(′), where N = A, C, G orU indicates 2′-0-methyl sugar modification; ′Chol′ stands forcholesterol conjugate and ′Cy-3′ stands for a Cy3 conjugate (Cy3 Quasarbuilding blocks were purchased from Biosearch Technologies, Novato,California).

In a preferred embodiment, the antisense strand of an iRNA agentconjugated to a lipophilic agent has the sequence of an antisense strandlisted in Table 1 or Table 2, or differs from an antisense strand listedin Table 1 or Table 2 by no more than 1, 2, 3, 4, or 5 nucleotides. Inanother preferred embodiment, the sense strand of an iRNA agentconjugated to a lipophilic agent has the sequence of an antisense strandlisted in Table 1 or Table 2, or differs from an antisense strand listedin Table 1 or Table 2 by no more than 1, 2, 3, 4, or 5 nucleotides. Inanother preferred embodiment, the antisense strand of the iRNA agent hasat least one modification described in Table 1 or Table 2 (e.g., acholesterol, 2′-OMe, phosphorothioate, or Cy-3 modification). In anotherpreferred embodiment, the antisense strand will have the modificationsshown in Table 1 or Table 2. The antisense strand of an iRNA agent canhave one or fewer modifications, e.g., the type shown in Table 1 orTable 2, or can have one or more additional modifications, e.g., thetype shown in Table 1 or Table 2. In another preferred embodiment, thesense strand of the iRNA agent has at least one modification describedin Table 1 or Table 2 (e.g., a cholesterol, 2′-OMe, phosphorothioate, orCy-3 modification). In another preferred embodiment, the sense strandwill have the modifications shown in Table 1 or Table 2. The sensestrand of an iRNA agent can have one or fewer modifications, e.g., thetype shown in Table 1 or Table 2, or can have one or more additionalmodifications, e.g., the type shown in Table 1 or Table 2.

In another embodiment, the iRNA agent targets an htt nucleic acid. Inone embodiment, the antisense strand of the iRNA agent includes anantisense sequence described herein, e.g., an antisense sequence listedin Table 1 or Table 2. In another embodiment, the sense strand of theiRNA agent includes the nucleotide sequence of a sense strand describedherein, e.g., a sense sequence listed in Table 1 or Table 2. In yetanother embodiment, the antisense strand of the iRNA agent overlaps anantisense sequence described herein, e.g., an antisense sequence listedin Table 1 or Table 2, e.g., by at least 1, 5, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides Likewise, the sensestrand of the iRNA agent overlaps a sense sequence described herein,e.g., a sense sequence listed in Table 1 or Table 2, e.g., by at least1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24nucleotides.

In a particularly preferred embodiment, the iRNA agent targets a nucleicacid involved in a neurological disease. The iRNA agent has an antisensestrand complementary to a nucleotide sequence of the target nucleicacid, and a sense strand sufficiently complementary to hybridize to theantisense strand. The iRNA agent also includes a liphophilic moiety thatfacilitates its uptake into a neural cell. In a preferred embodiment,the lipophilic moiety is a cholesterol. In another embodiment, the iRNAagent includes a modification that improves the stability ordistribution of the iRNA agent in a biological sample. The iRNA agentscan further be in isolated form or can be part of a pharmaceuticalcomposition used for the methods described herein, particularly as apharmaceutical composition formulated for delivery to a neural cell orformulated for parental administration. The pharmaceutical compositionscan contain one or more iRNA agents, and in some embodiments, willcontain two or more iRNA agents. In one embodiment, the iRNA agentincludes a 2′-modified nucleotide, e.g., a 2′-O-methylated nucleotide.In another embodiment, the iRNA agent includes a phosphorothioate.

In another embodiment, the iRNA agent targets a wildtype nucleic acid,e.g., a wildtype htt RNA, involved in the pathogenesis of a neurologicaldisorder, and in yet another embodiment, the iRNA agent targets apolymorphism or mutation of the nucleic acid. In certain embodiments,the iRNA agent can target a sequence in a codon of the open readingframe, the 3′UTR or the 5′UTR of the mRNA transcript of the geneinvolved in the neurological disorder. In one embodiment, the iRNA agenttargets a spliced isoform of mRNA.

In one embodiment, the human carries a form of the huntingtin gene thatincludes an expanded CAG trinucleotide repeat, i.e., more than 30 CAGtrinucleotide repeats (e.g., 35, 40, 50, 60, 70, 80, 90, 100 or more CAGtrinucleotide repeats), which results in an abnormal form of thehuntingtin polypeptide including an expansion of the polypeptide'snormal polyglutamine tract. In another embodiment, the human isdiagnosed with Huntington's Disease (HD). In one embodiment, the humancarries a polymorphism or mutation in the huntingtin gene. For example,the human can carry a polymorphism at position 171, e.g., an A171Cpolymorphism, in the huntingtin gene according to the sequence numberingin GenBank Accession No. NM_(—)002111 (Aug. 8, 2005). In anotherembodiment, the iRNA agent targets a nucleic acid that encodes apolypeptide known to interact with the huntingtin protein. For example,the iRNA agent can target a Huntingtin-associated protein-1 (HAP-1)nucleic acid.

In a preferred embodiment, the iRNA agent modified for enhanced uptakeinto neural cells does not target an alpha-synuclein (SNCA) RNA.

In another embodiment, the iRNA agent modified for enhanced uptake intoneural cells, e.g., conjugated to a cholesterol, is at least 21nucleotides long and includes a sense RNA strand and an antisense RNAstrand, wherein the antisense RNA strand is 25 or fewer nucleotides inlength, and the duplex region of the iRNA agent is 18-25 nucleotides inlength. The iRNA agent may further include a nucleotide overhang having1 to 4 unpaired nucleotides, and the unpaired nucleotides may have atleast one phosphorothioate dinucleotide linkage. The nucleotide overhangcan be, e.g., at the 3′ end of the antisense strand of the iRNA agent.

In one aspect, the invention features a method of down regulatingexpression of a target gene in a neural cell. In one embodiment themethod includes contacting an iRNA agent with the neural cell for a timesufficient to allow uptake of the iRNA agent into the cell. In anotherembodiment, the iRNA agent includes a sense strand and an antisensestrand that form an RNA duplex. The iRNA agent also comprises alipophilic moiety, e.g., a cholesterol, and the antisense strand of theiRNA agent comprises a nucleotide sequence sufficiently complementary toa target sequence of about 18 to 25 nucleotides of an RNA expressed fromthe target gene. In a preferred embodiment, the lipophilic moiety isconjugated to at least one end of the sense strand, e.g., to the 3′ endof the sense strand. In another embodiment, the sense strand and theantisense strand have a sequence selected from the sense and antisensestrands listed in Table 1 or Table 2.

In another aspect, the invention features a method of treating a humanthat includes identifying a human diagnosed as having or at risk fordeveloping a neurological disorder, and administering to the human aniRNA agent that targets a gene expressed in a neural cell. In oneembodiment, expression of the gene is associated with symptoms of theneurological disorder. In another embodiment, the iRNA agent includes asense strand and an antisense strand that form an RNA duplex, and theiRNA agent includes a lipophilic moiety, e.g., a cholesterol. In anotherembodiment, the antisense strand of the iRNA agent includes a nucleotidesequence sufficiently complementary to a target sequence of about 18 to25 nucleotides of an RNA expressed from the target gene. In a preferredembodiment, the lipophilic moiety is conjugated to at least one end ofthe sense strand, e.g., to the 3′ end of the sense strand, and inanother embodiment, the iRNA agent includes a phosphorothioate or a 2′modification, e.g., a 2′OMe or 2′O-fluoro modification. In oneembodiment, the sense and antisense strands include a sequence selectedfrom the sense and antisense strands listed in Table 1 or Table 2.

In one embodiment, the antisense strand of the iRNA agent includes asequence complementary to a polymorphism of an htt RNA. In anotherembodiment, the human has or is at risk for developing Huntington'sdisease. In another embodiment, the human carries a genetic variation ina Parkin gene or a ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene,and the human has or is at risk for developing Parkinson's disease. Inanother embodiment, the human has or is at risk for developingAlzheimer's Disease, multiple system atrophy, or Lewy body dementia.

In another aspect, the invention features a pharmaceutical compositionincluding an iRNA agent conjugated to a lipophilic moiety for enhanceduptake into neural cells, e.g., conjugated to a cholesterol molecule,and a pharmaceutically acceptable carrier. Preferably, the iRNA agenttargets a nucleic acid involved in a neurological disease or disorder.

In a particularly preferred embodiment, the pharmaceutical compositionincludes an iRNA agent targeting an htt nucleic acid and apharmaceutically acceptable carrier. The iRNA agent has an antisensestrand complementary to a nucleotide sequence of an htt RNA, and a sensestrand sufficiently complementary to hybridize to the antisense strand.In one embodiment, the iRNA agent includes a lipophilic moiety thatfacilitates its uptake into a neural cell. In one embodiment, thelipophilic moiety is a ligand that includes a cationic group. In anotherembodiment, the lipophilic moiety is attached to one or both ends of oneor both strands of the iRNA agent. In a preferred embodiment, thelipophilic moiety is attached to one end of the sense strand of the iRNAagent, and in another preferred embodiment, the ligand is attached tothe 3′ end of the sense strand. In certain embodiments, the lipophilicagent is, e.g, cholesterol, vitamin E, vitamin K, vitamin A, folic acidor a cationic dye, such as Cy3. In a preferred embodiment, thelipophilic moiety is a cholesterol.

In another embodiment, the iRNA agent of the pharmaceutical compositionincludes a modification that improves the stability or distribution ofthe iRNA agent in a biological sample. The iRNA agents can further be inisolated form or can be part of a pharmaceutical composition used forthe methods described herein, particularly as a pharmaceuticalcomposition formulated for delivery to a neural cell or formulated forparental administration. The pharmaceutical compositions can contain oneor more iRNA agents, and in some embodiments, will contain two or moreiRNA agents. In one embodiment, the iRNA agent includes a 2′-modifiednucleotide, e.g., a 2′-O-methylated nucleotide. In another embodimentthe iRNA agent includes a phosphorothioate.

In a particularly preferred embodiment, htt RNA levels in a neural cellare reduced by contacting the neural cell of the subject with an iRNAagent modified for enhanced uptake into neural cells. In a preferredembodiment, the ligand is a cholesterol.

In another aspect, the invention features a method of making an iRNAagent that targets a nucleic acid expressed in neural cells and that ismodified for enhanced uptake into neural cells. The method includesselecting a nucleotide sequence of between 18 and 25 nucleotides longfrom the nucleotide sequence of a target mRNA, e.g., an htt mRNA, andsynthesizing the iRNA agent. The sense strand of the iRNA agent includesthe nucleotide sequence selected from the target RNA, and the antisensestrand is sufficiently complementary to hybridize to the sense strand.The method includes incorporating at least one lipophilic moiety intothe iRNA agent, e.g., onto at least one end of the sense strand of theiRNA agent. In a preferred embodiment, the lipophilic moiety isincorporated onto the 3′ end of the sense strand of the iRNA agent. Inone embodiment, a cationic dye, e.g., Cy3, is incorporated into at leastone strand of the iRNA agent, e.g., on the 3′ or 5′ end of the iRNAagent. In one embodiment, more than one lipophilic moiety, e.g., morethan one different kind of lipophilic moiety is incorporated into theiRNA agent. In certain embodiments, the iRNA agent includes the ligandconjugates illustrated in Table 1 or Table 2. In other embodiments themethod of making the iRNA agent includes use of the building blocksillustrated in Table 1 or Table 2. In yet other embodiments, the methodsfeatured in the invention include methods of making the iRNA agentslisted in Table 1 or Table 2, which target htt RNA. In one embodiment,the method further includes administering the iRNA agent to a subject,e.g., a mammalian subject, such as a human subject, such as a humanhaving or at risk for developing a neurological disease or disorder. Inone embodiment, the human has or is at risk for developing HD.

In another aspect, the invention features a method of evaluating an iRNAagent, e.g., an iRNA agent conjugated with a lipophilic agent forenhanced uptake into neural cells. The method includes: providing acandidate iRNA agent modified for enhanced uptake into neural cells,e.g., conjugated with a cholesterol molecule and determining whether theiRNA agent is taken up into neural cells. In one embodiment, the iRNAagent is conjugated with a detectable marker, e.g., a fluorescentmarker, such as Cy3 or Cy5, and uptake into the cell is assayed byfluorescence. In another embodiment e.g., by the use of one or more ofthe test systems described herein, if said candidate agent modulates,e.g., inhibits, target gene expression.

In a preferred embodiment the method includes evaluating the iRNA agentin a first test system; and, if a predetermined level of gene expressionis observed, evaluating the candidate in a second, preferably different,test system. In a particularly preferred embodiment the second testsystem includes administering the candidate iRNA agent to a neural cellof an animal and evaluating the effect of the candidate agent on targetgene expression in the animal.

A test system can include: contacting the candidate iRNA agent with atarget nucleic acid, e.g., a nucleic acid expressed in neural cells,such as an htt RNA. The iRNA is preferably contacted with the targetnucleic acid in vitro, and it is determined whether there is aninteraction, e.g., binding of the candidate agent to the target. Thetest system can include contacting the candidate agent with a neuralcell and evaluating modulation of neural gene expression, e.g., htt geneexpression. For example, this can include contacting the candidate iRNAagent with a neural cell capable of expressing htt RNA (from anendogenous gene or from an exogenous construct) and evaluating the levelof htt or htt RNA. In a preferred embodiment, the candidate iRNA agentincludes a modification that enhances uptake of the candidate iRNA agentinto the neural cell. In a particularly preferred embodiment, themodification is a cholesterol molecule, e.g., a cholesterol attached tothe sense strand of the iRNA agent, e.g., to the 3′ end of the sensestrand. In another embodiment the test system can include contacting thecandidate agent with a cell which expresses an RNA or protein from aportion of the neural gene linked to a heterologous sequence, e.g., amarker protein, e.g., a fluorescent protein such as GFP, which constructcan be either chromosomal or episomal, and determining the effect on RNAor protein levels. The test system can also include contacting thecandidate iRNA agent, in vitro, with a tissue sample, e.g., a braintissue sample, e.g., a slice or section, an optical tissue sample, orother sample which includes neural tissue, and evaluating the level ofneural polypeptide or RNA, e.g., htt polypeptide or RNA. The test systemcan include administering the candidate iRNA agent, in vivo, to ananimal, and evaluating the level of neural polypeptide or neural RNA,e.g., htt polypeptide or htt RNA. In any of these, the effect of thecandidate agent on neural gene expression can include comparing geneexpression with a predetermined standard, e.g., with control, e.g., anuntreated cell, tissue or animal. Gene expression can be compared, e.g.,before and after contacting with the candidate iRNA agent. The methodallows determining whether the iRNA agent is useful for inhibiting httgene expression.

A “neural gene” is a gene expressed in neural cells. A neural gene canbe expressed exclusively in neural cells, or can be expressed in othercell types in addition to the neural cell.

In one embodiment, neural gene expression can be evaluated by a methodto examine neural RNA levels (e.g., Northern blot analysis, RT-PCR, orRNAse protection assay) or neural polypeptide levels (e.g., Westernblot, immunohistochemistry, or autofluorescence assays (e.g., to detectGFP or luciferase expression)).

A “neural cell” is a cell of the nervous system, e.g., the peripheral orthe central nervous system. A neural cell can be a nerve cell (i.e., aneuron), e.g., a sensory neuron or a motoneuron, or a glial cell.Exemplary neurons include dorsal root ganglia of the spinal cord, spinalmotor neurons, retinal bipolar cells, cortical and striatal cells of thebrain, hippocampal pyramidal cells, and purkinje cells of thecerebellum. Exemplary glial cells include oligodendrocytes andastrocytes of the central nervous system, and the Schwann cells of theperipheral nervous system.

By “enhanced uptake into neural cells” is meant that higher levels of amodified iRNA agent are incorporated into a neural cell than unmodifiediRNA agent when the cells exposed to each type of iRNA agent are treatedunder similar conditions, in in vitro or in vivo conditions.

In one embodiment, e.g., as a second test, the agent is administered toan animal, e.g., a mammal, such as a mouse, rat, rabbit, human, ornon-human primate, and the animal is monitored for an effect of theagent. For example, a tissue of the animal, e.g., a brain tissue, isexamined for an effect of the agent on neural gene expression. Thetissue can be examined for the presence of neural RNA and/or proteinlevels, for example. In one embodiment, the animal is observed tomonitor an improvement or stabilization of a cognitive symptom. Theagent can be administered to the animal by any method, e.g., orally, orby intrathecal or parenchymal injection, such as by stereoscopicinjection into the brain.

In a particularly preferred embodiment, the invention features a methodof evaluating an iRNA agent, e.g., an iRNA agent described herein, thattargets a nucleic acid expressed in neural cells, e.g., an htt nucleicacid. The method includes providing an iRNA agent that targets a nucleicacid expressed in neural cells (e.g., an htt RNA); contacting the iRNAagent with a neural cell containing and capable of expressing thenucleic acid; and evaluating the effect of the iRNA agent on expressionof the nucleic acid, e.g., by comparing gene expression with a control,e.g., in the cell. Gene expression can be compared, e.g., before andafter contacting the iRNA agent with the cell. The method allowsdetermining whether the iRNA agent is useful for inhibiting geneexpression. For example, the iRNA agent can be determined to be usefulfor inhibiting neural gene expression if the iRNA agent reducesexpression by a predetermined amount, e.g., by 10, 25, 50, 75, or 90%,e.g., as compared with a predetermined reference value, e.g., ascompared with the amount of neural RNA or protein prior to contactingthe iRNA agent with the cell. The neural gene can be endogenously orexogenously expressed.

The methods and compositions featured in the invention, e.g., themethods and iRNA compositions to treat the neurological disordersdescribed herein, can be used with any dosage and/or formulationdescribed herein, as well as with any route of administration describedherein.

Thus, in another aspect, the invention features a method of treating asubject by administering an agent which inhibits the expression of agene in a neural cell. In a preferred embodiment, the subject is amammal, such as a human, e.g., a subject diagnosed as having, or at riskfor developing a neurological disease or disorder. Agents that inhibitneural gene expression include iRNA agents and antisense molecules thattarget htt RNA.

A “substantially identical” sequence includes a region of sufficienthomology to the target gene, and is of sufficient length in terms ofnucleotides, that the iRNA agent (e.g., the iRNA agent conjugated to alipophilic moiety for enhanced uptake into neural cells), or a fragmentthereof, can mediate down regulation of the target gene. A sequence ofthe iRNA agent that is substantially identical to a target RNA istypically a sequence on the sense strand of a double stranded iRNAagent.

A “substantially complementary” sequence includes a region of sufficientcomplementarity to the target gene, and is of sufficient length in termsof nucleotides, that the iRNA agent (e.g., the iRNA agent conjugated toa lipophilic moiety for enhanced uptake into neural cells), or afragment thereof, can mediate down regulation of the target gene. Asequence of the iRNA agent that is substantially complementary to atarget RNA is typically a sequence on the antisense strand of a doublestranded iRNA agent. Thus, the iRNA agent is or includes a region whichis at least partially, and in some embodiments fully, complementary to atarget RNA transcript, e.g, an RNA transcript expressed in a neuralcell. It is not necessary that there be perfect complementarity betweenthe iRNA agent and the target, but the correspondence must be sufficientto enable the iRNA agent, or a cleavage product thereof, to directsequence specific silencing, e.g., by RNAi cleavage of the target RNA,e.g., mRNA. Complementarity, or degree of homology with the targetstrand, is most critical in the antisense strand. While perfectcomplementarity, particularly in the antisense strand, is often desiredsome embodiments can include, particularly in the antisense strand, oneor more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respectto the target RNA). The mismatches, particularly in the antisensestrand, are most tolerated in the terminal regions and if present arepreferably in a terminal region or regions, e.g., within 6, 5, 4, or 3nucleotides of the 5′ and/or 3′ terminus. The sense strand need only besufficiently complementary with the antisense strand to maintain theoverall double strand character of the molecule.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogates, which are described herein or are well known inthe RNA synthetic art. While numerous modified RNAs and nucleosidesurrogates are described, preferred examples include those which havegreater resistance to nuclease degradation than do unmodified RNAs.Preferred examples include those that have a 2′ sugar modification, amodification in a single strand overhang, preferably a 3′ single strandoverhang, or, particularly if single stranded, a 5′ modification whichincludes one or more phosphate groups or one or more analogs of aphosphate group.

An “iRNA agent” (abbreviation for “interfering RNA agent”) as usedherein, is an RNA agent, which can downregulate the expression of atarget gene, preferably an endogenous or pathogen target RNA expressedin a neural cell. While not wishing to be bound by theory, an iRNA agentmay act by one or more of a number of mechanisms, includingpost-transcriptional cleavage of a target mRNA sometimes referred to inthe art as RNAi, or pre-transcriptional or pre-translational mechanisms.An iRNA agent is preferably a double stranded (ds) iRNA agent.

Further, one aspect of the invention pertains to a method ofdownregulating expression target gene in a neural cell distal to thesite of administration, the method comprising contacting an iRNA agentwith the neural cell for a time sufficient to allow uptake of the iRNAagent into the cell, wherein (i) the iRNA agent comprises a sense and anantisense strand that form an RNA duplex, and (ii) the sequence of theantisense strand of the iRNA agent comprises a nucleotide sequencesufficiently complementary to a target sequence of about 18 to 25nucleotides of an RNA expressed from the target gene.

In another aspect, the invention pertains to a method of downregulatingexpression of a target gene in a neural cell, the method comprisingcontacting an iRNA agent with the neural cell for a time sufficient toallow uptake of the iRNA agent into the cell, wherein (i) the iRNA agentcomprises a sense and an antisense strand that form an RNA duplex, (ii)the iRNA agent comprises a lipophilic moiety, and (iii) the sequence ofthe antisense strand of the iRNA agent comprises a nucleotide sequencesufficiently complementary to a target sequence of about 18 to 25nucleotides of an RNA expressed from the target gene.

In one embodiment, the cells are contacted for a time sufficient toallow axonal transport of said iRNA.

In one embodiment, the iRNA agent comprises a lipophilic moiety.

In one embodiment, the lipophilic moiety is a cholesterol.

In one embodiment, the lipophilic moiety is conjugated to the sensestrand.

In one embodiment, the lipophilic moiety is conjugated to the 3′ end ofthe sense strand.

In one embodiment, the antisense sequence differs by no more than fournucleotides from an antisense sequence listed in Table 1.

In one embodiment, the antisense strand is selected from an antisensestrand listed in Table 1.

In one embodiment, the iRNA agent further comprises a phosphorothioateor a 2′-OMe modification.

In one embodiment, the iRNA agent is provided in a solution that lacks atransfection reagent.

In one embodiment, the iRNA agent is provided in a solution comprising atransfection reagent.

In another aspect, the invention pertains to a method of treating ahuman comprising identifying a human having or at risk for developing aneurological disorder, the method comprising administering to the humanan iRNA agent that targets a gene expressed in a neural cell distal tothe site of administration, wherein the expression of the gene isassociated with symptoms of the neurological disorder, and wherein (i)the iRNA agent comprises a sense and an antisense strand that form anRNA duplex, and (ii) the antisense strand of the iRNA agent comprises anucleotide sequence sufficiently complementary to a target sequence ofabout 18 to 25 nucleotides of an RNA expressed from the target gene.

In another aspect, the invention pertains to a method of treating ahuman comprising identifying a human having or at risk for developing aneurological disorder, and administering to the human an iRNA agent thattargets a gene expressed in a neural cell, wherein the expression of thegene is associated with symptoms of the neurological disorder, andwherein (i) the iRNA agent comprises a sense and an antisense strandthat form an RNA duplex, (ii) the iRNA agent comprises a lipophilicmoiety, and (iii) the antisense strand of the iRNA agent comprises anucleotide sequence sufficiently complementary to a target sequence ofabout 18 to 25 nucleotides of an RNA expressed from the target gene.

In one embodiment, the iRNA agent comprises a lipophilic moiety.

In one embodiment, the lipophilic moiety is a cholesterol,

In one embodiment, the lipophilic moiety is conjugated to the sensestrand.

In one embodiment, the lipophilic moiety is conjugated to the 3′ end ofthe sense strand.

In one embodiment, the iRNA agent further comprises a phosphorothioateor a 2′-OMe modification.

In one embodiment, the antisense sequence differs by no more than fournucleotides from an antiisense sequence listed in Table 1.

In one embodiment, the antisense strand is selected from an antisensestrand listed in Table 1.

In one embodiment, the antisense strand of the iRNA agent comprises asequence complementary to a sequence comprising a polymorphism of ahuntingtin (htt) RNA.

In one embodiment, the human carries a genetic variation in a Parkingene or a ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene.

In one embodiment, the neurological disorder is Huntington's disease.

In one embodiment, the neurological disorder is Parkinson's disease.

In one embodiment, the neurological disorder is Alzheimer's Disease,multiple system atrophy, or Lewy body dementia,

In one embodiment, the iRNA agent comprises a nucleotide overhang having1 to 4 unpaired nucleotides.

In one embodiment, the iRNA agent is provided in a solution that lacks atransfection reagent.

In one embodiment, the iRNA agent is provided in a solution comprising atransfection reagent.

In one embodiment, the iRNA agent is administered as a sustained doseformulation.

In one embodiment, the iRNA agent is administered in multiple doses overa prolonged time period.

In one embodiment, the iRNA agent is administered as a single dose.

In one embodiment, the administration of a second iRNA agent, wherein(i) the second iRNA agent comprises a sense and an antisense strand thatform an RNA duplex, and (ii) the antisense strand of the iRNA agentcomprises a nucleotide sequence sufficiently complementary to a secondtarget sequence of about 18 to 25 nucleotides of the RNA expressed fromthe target gene.

In another aspect, the invention pertains to a method of reducing theamount of huntingtin (htt) RNA in a neural cell of a subject,comprising: contacting the neural cell with an iRNA agent, wherein saidneural cell is distal to the site of action and the iRNA agent comprisesa sense and an antisense strand, wherein the sense and the antisensestrands form an RNA duplex, wherein the antisense strand comprises anucleotide sequence that differs by no more than four nucleotides froman antisense sequence listed in Table 1.

In one embodiment, the iRNA agent further comprises a lipophilic moiety.

In one embodiment, the cells are contacted for a time sufficient toallow uptake of the iRNA agent into the cells and axonal transport ofsaid iRNA

In one embodiment, the iRNA agent further comprises a phosphorothioateor a 2′-OMe modification.

In one embodiment, the iRNA agent comprises an antisense strandcomprising a sequence selected from the antisense strands listed inTable 1.

In one embodiment, the iRNA agent comprises a sense strand selected fromthe sense strands listed in Table 1.

In one embodiment, the iRNA agent comprises an antisense strandcomprising a sequence complementary to sequence comprising apolymorphism of an htt RNA.

In one embodiment, the polymorphism is an A to C at position 171according to the sequence of GenBank Accession No. NM_(—)002111.

In one embodiment, the iRNA agent comprises a nucleotide overhang having1 to 4 unpaired nucleotides.

In another aspect, the invention pertains to an isolated iRNA agentcomprising a sense and an antisense strand, wherein the sense and theantisense strands form an RNA duplex, wherein the antisense strandcomprises a nucleotide sequence that differs by no more than fournucleotides from an antisense sequence listed in Table 1, and whereinthe iRNA agent comprises a lipophilic moiety.

In one embodiment, the lipophilic moiety is a cholesterol molecule.

In one embodiment, the lipophilic moiety is attached to the sensestrand.

In one embodiment, the lipophilic moiety is attached to the 3′ end ofthe sense strand.

In one embodiment, the iRNA agent further comprises a phosphorothioatemodification, or a 2′-OMe modification.

In one embodiment, the antisense strand comprises a sequence selectedfrom the antisense sequences listed in Table 1.

In one embodiment, the sense strand of the iRNA agent comprises asequence selected from the sense sequences listed in Table 1.

In one embodiment, the iRNA agent is at least 21 nucleotides in length,and the duplex region of the iRNA agent is about 18-25 nucleotides inlength.

In one embodiment, the iRNA agent comprises a nucleotide overhang having1 to 4 unpaired nucleotides.

In another aspect, the invention pertains to a pharmaceuticalcomposition, comprising (i) an iRNA agent comprising a sense and anantisense strand, wherein the sense and the antisense strands form anRNA duplex, wherein the antisense strand comprises a nucleotide sequencethat differs by no more than four nucleotides from an antisense sequencelisted in Table 1, and wherein the iRNA agent comprises a lipophilicmoiety, and

(ii) a pharmaceutically acceptable carrier.

In one embodiment, the iRNA agent further comprises a phosphorothioateor a 2′-OMe modification.

In one embodiment, the antisense strand of the iRNA agent comprises asequence selected from the antisense sequences listed in Table 1.

In one embodiment, the sense strand of the iRNA agent comprises asequence from the selected from the sense sequences listed in Table 1.

In one embodiment, the iRNA agent comprises a nucleotide overhang having1 to 4 unpaired nucleotides.

In another aspect, the invention pertains to a method of evaluating aniRNA agent for enhanced uptake into neural cells comprising: providing acandidate iRNA agent conjugated to a lipophilic agent, wherein the iRNAagent is in a solution that does not contain a transfection reagent,contacting the iRNA agent with a neural cell for a time sufficient foruptake into the neural cell and determining if the iRNA agent is takenup by the neural cell.

In one embodiment, the method includes a step of evaluating the iRNAagent in a cell culture system; and, if a predetermined level of uptakeinto the neural cell is observed, evaluating the candidate in an animal.

In one embodiment, the iRNA agent comprises an antisense strand that issubstantially complementary to a target RNA in the neural cell, and themethod further comprises determining whether the candidate iRNA agentdecreases expression of a target RNA in the neural cell.

In one embodiment, the lipophilic agent is a cholesterol.

In one embodiment, the target RNA is a huntingtin RNA.

In one embodiment, the iRNA agent comprises an antisense strand that issubstantially complementary to a target RNA in the neural cell, and themethod further comprises determining whether the candidate iRNA agentdecreases expression of the target RNA in a neural cell of the animal.

In one embodiment, the target RNA is a huntingtin RNA.

In one embodiment, the animal is monitored for an effect of the iRNAagent.

In one embodiment, brain tissue from the animal is examined for aneffect of the iRNA agent on target gene expression.

In one embodiment, the determining step comprises performing a methodselected from the group consisting of Northern blot, Western blot,RT-PCR, and RNAse protection assay.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thisdescription, and from the claims. This application incorporates allcited references, patents, and patent applications by references intheir entirety for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1D are images of in situ staining for pathological hallmarks ofHuntington's disease found in mice treated with lentivirus-mut-htt.Staining was by immunohistochemistry against htt protein. FIG. 1A is anin situ stain of striatum tissue. FIGS. 1B, 1C and 1D are in situ stainsof the striatum near the globus pallidus. Scale bar=25 μm.

FIG. 2. Intrastriatal injection of cholesterol-conjugated dsRNAAL-DP-1799 targeting huntingtin changes pattern of cellularhuntingtin-immunoreactivity in a mouse model of Huntington's disease.

FIG. 3. Intrastriatal injection of cholesterol-conjugated dsRNAAL-DP-1799 targeting huntingtin reduces size ofhuntingtin-immunoreactive inclusions in cortex and striatum, and reducesneuropil aggregates in a mouse model of Huntington's disease.

FIG. 4. Intrastriatal injection of cholesterol-conjugated dsRNAAL-DP-1799 targeting huntingtin increases the number ofhuntingtin-immunoreactive cells in striatum in a mouse model ofHuntington's disease.

FIG. 5. Intrastriatal injection of cholesterol-conjugated dsRNAAL-DP-1799 targeting huntingtin reduces abnormal clasping behavior in amouse model of Huntington's disease.

DETAILED DESCRIPTION

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNAthrough a process known as RNA interference (RNAi). The process occursin a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA aresequence-specific mediators of RNA silencing, e.g., by causing RNAdegradation. While not wishing to be bound by theory, it may be that amolecular signal, which may be merely the specific length of thefragments, present in these 21-23 nt fragments, recruits cellularfactors that mediate RNAi. Described herein are methods for preparingand administering these 21-23 nt fragments, and other iRNA agents, andtheir use for specifically inactivating gene function in neural cells.The use of iRNA agents (or recombinantly produced or chemicallysynthesized oligonucleotides of the same or similar nature) enables thetargeting of specific mRNAs for silencing in mammalian cells. Inaddition, longer dsRNA agent fragments can also be used, e.g., asdescribed below.

Although, in mammalian cells, long dsRNAs can induce the interferonresponse which is frequently deleterious, short dsRNAs (sRNAs) do nottrigger the interferon response, at least not to an extent that isdeleterious to the cell and host. In particular, the length of the iRNAagent strands in an sRNA agent can be less than 31, 30, 28, 25, or 23nt, e.g., sufficiently short to avoid inducing a deleterious interferonresponse. Thus, the administration of a composition of sRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of a target gene while circumventing the interferonresponse. Further, use of a discrete species of iRNA agent can be usedto selectively target one allele of a target gene, e.g., in a subjectheterozygous for the allele.

Moreover, in one embodiment, a mammalian cell is treated with an iRNAagent that disrupts a component of the interferon response, e.g.,dsRNA-activated protein kinase PKR. Such a cell can be treated with asecond iRNA agent that includes a sequence complementary to a target RNAand that has a length that might otherwise trigger the interferonresponse.

In a typical embodiment, the subject is a mammal such as a cow, horse,mouse, rat, dog, pig, goat, or a primate. The subject can be a dairymammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken,turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, thesubject is a human, e.g., a normal individual or an individual that has,is diagnosed with, or is predicted to have a neurological disease ordisorder.

A neurological disease or disorder is any disease or disorder thataffects the nervous system (the central or peripheral nervous system).Exemplary neurological diseases and disorders include Huntingtons'sDisease (HD), Parkinson's Disease (PD), Amyotropic Lateral Sclerosis(ALS), Alzheimer's Disease, Lewy body dementia, Multiple System Atrophy,spinal and bulbar muscular atrophy (Kennedy's disease), TouretteSyndrome, Autosomal dominant spinocerebellar ataxia (SCA) (e.g., Type 1SCA1, Type 2 SCA2, Type 3 (Machado-Joseph disease) SCA3/MJD, Type 6SCA6, Type 7 SCAT, Type 8 SCA8, Friedreich's Ataxia and Dentatorubralpallidoluysian atrophy DRPLA/Haw-River syndrome), schizophrenia, ageassociated memory impairment, autism, attention-deficit disorder,bipolar disorder, and depression.

Because oligonucleotide agent-mediated modulation persists for severaldays after administering the oligonucleotide agent composition, in manyinstances it is possible to administer the composition with a frequencyof less than once per day, or, for some instances, only once for theentire therapeutic regimen. For example, treatment of some cancerousneural cells may be mediated by a single bolus administration, whereas achronic viral infection may require regular administration, e.g., onceper week or once per month. For example, treatment of an astrocytoma maybe treated with a single bolus administration of an iRNA agentconjugated to a lipophilic agent.

Treatment of Neurological Diseases and Disorders

Any patient having a neurological disease or disorder is a candidate fortreatment with a method or composition described herein. Presymptomaticsubjects can also be candidates for treatment with an iRNA agenttargeted to neural cells. For example, a presymptomatic human determinedto be at risk for HD is a candidate for treatment with an anti-htt iRNAagent conjugated to a lipophilic molecule, e.g., a cholesterol molecule,for delivery to neural cells. In one embodiment, a presymptomaticcandidate is identified by either or both of risk-factor profiling andfunctional neuroimaging (e.g., by fluorodopa and positron emissiontomography). For example, the candidate subject can be identified byrisk-factor profiling followed by functional neuroimaging.

Individuals having a particular genotype are candidates for treatment.In some embodiments the patient will carry a particular genetic mutationthat places the patient at increased risk for developing a disorder ofthe nervous system, e.g., HD. For example, an individual carrying a CAGtrinucleotide expansion in htt (e.g., more than 36 repeats) is atincreased risk for developing HD and is a candidate for treatment withan iRNA agent featured in the invention, e.g., conjugated to acholesterol molecule for enhanced uptake into neural cells. The iRNAagent preferably targets the htt gene. In addition, a SNP in the httgene has been found to be an indicator of the presence of the expandedCAG repeat that triggers HD. The SNP is an A to C polymorphism atposition 171, according to the numbering of GenBank Accession No.NM_(—)002111. A human carrying this SNP is therefore a candidate fortreatment with an iRNA agent featured in the invention, or is at least acandidate for further genetic studies (such as for testing for the CAGrepeat expansion) which will further determine if the human is acandidate for treatment with an iRNA agent targeting htt and modifiedfor enhanced delivery to neurons.

In another example, the non-genetic risk factors for PD include age(e.g., over age 30, 35, 40, 45, or 50 years), gender (men are generallyhave a higher risk than women), pesticide exposure, heavy metalexposure, and head trauma. In general, exogenous and endogenous factorsthat disrupt the ubiquitin proteasomal pathway or more specificallyinhibit the proteasome, or which disrupt mitochondrial function, orwhich yield oxidative stress can increase the risk of an individual fordeveloping PD, and can contribute to the pathogenesis of PD. Thesefactors can be considered when evaluating the risk profile of acandidate subject for treatment with an iRNA agent modified for enhanceduptake into a neural cell, e.g., conjugated to a cholesterol molecule.

Design and Selection of iRNA Agents

Candidate iRNA agents can be designed by performing, for example, a genewalk analysis. Overlapping, adjacent, or closely spaced candidate agentscorresponding to all or some of the transcribed region can be generatedand tested. Each of the iRNA agents can be tested and evaluated for theability to down regulate target gene expression (see below, “Evaluationof Candidate iRNA agents”).

An iRNA agent can be rationally designed based on sequence informationand desired characteristics. For example, an iRNA agent can be designedaccording to the relative melting temperature of the candidate duplex.Generally, the duplex will have a lower melting temperature at the 5′end of the antisense strand than at the 3′ end of the antisense strand.This and other elements of rational design are discussed in greaterdetail below (see, e.g., sections labeled “Palindromes,” “Asymmetry,”and “Z-X-Y,” and “Differential Modification of Terminal DuplexStability” and “Other-than-Watson-Crick Pairing.”

Evaluation of Candidate iRNA Agents

A candidate iRNA agent, e.g., a candidate iRNA agent conjugated to alipophilic moiety, can be evaluated for its ability to be taken up intoneural cells. For example, a candidate iRNA agent conjugated to alipophilic moiety is provided in a solution that does not contain anadditional lipophilic moiety or transfection reagent to facilitateuptake into the cell. “Transfection reagents” include ions or othersubstances which substantially alter cell permeability to anoligonucleotide agent. Exemplary transfecting agents includeLipofectamine™ (Invitrogen, Carlsbad, Calif.), Lipofectamine 2000™,TransIT-TKO™ (Minis, Madison, Wis.), FuGENE 6 (Roche, Indianapolis,Ind.), polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.),DOTAP, DOSPER, Metafectene™ (Biontex, Munich, Germany), or anothertransfection reagent.

The candidate iRNA agent conjugated to a lipophilic moiety can beevaluated in a cell culture system, such as in a neural cell culture. Ifa predetermined level of uptake into neural cells is observed, thecandidate iRNA agent can be evaluating in an animal, e.g., in a mouse,rat, rabbit, dog, cat, or monkey. The cell cultures can be mammaliancell cultures, such as mouse, rat or human cell cultures. Exemplaryneural cell cultures include cortical cell lines, striatal cell lines(e.g., ST14A), pheocromocytoma cell lines (e.g., PC12), neuroblastomacell lines (e.g., N2a), and the like. The cell cultures can be, forexample, non-tumor- or tumor-derived neuronal cell lines, and can bederived from, for example, a glioma, glioblastoma, meduloblastoma,retinoblastoma, or a neuroendocrine cell line. Exemplary cell lines canbe provided by the American Type Culture Collection (ATCC) (Manassus,Va.).

A candidate iRNA agent that includes a lipophilic moiety, e.g., acholesterol, can include an antisense strand that is substantiallycomplementary to a target RNA in the neural cell, e.g., an htt RNA, andthe method of evaluating the iRNA agent can include determining whetherthe agent decreases expression of the target RNA in the cell.

A candidate iRNA agent that can be taken up into neural cells can befurther tested for uptake into a neural cell in vivo. For example,following administration of the iRNA agent, such as by direct injectioninto the animal, e.g., into the brain of the animal, the tissue at thesite of injection can be examined for uptake of the iRNA agent.Detection of the iRNA agent can be accomplished by a variety of methods.For example, if the iRNA agent is labeled with a fluorescent molecule,such as Cy3, Cy5, rhodamine or FITC label, uptake of the iRNA agent canbe assayed by monitoring for the uptake of the fluorescent label.Detection can also be accomplished by in situ hybridization with anoligonucleotide probe, e.g., to detect the presence of the iRNA. Assaysto detect target gene product (target RNA or protein) can also be usedto monitor uptake of the candidate iRNA agent into neural cells.Detection can be, for example, by in situ hybridization with anoligonucleotide probe to detect the target RNA or byimmunohistochemistry techniques to detect the target polypeptide.Alternatively, target RNA or protein can be isolated from the tissuecontaining the neural cells, and the RNA detected by, e.g., Northernblot, RT-PCR, or RNAse protection assay, or the target protein detectedby Western blot analysis. If a decreased level of RNA or protein isdetected, it can be determined that a sufficient amount of iRNA agent isentering the cell to cause the observed decrease in RNA or proteinexpression.

For example, a candidate iRNA agent conjugated with a lipophilic moietycan be provided, and contacted with a neural cell, e.g., a neural cellthat expresses a target gene, such as the htt gene. The level of targetgene expression prior to and following contact with the candidate iRNAagent can be compared. The target gene can be an endogenous or exogenousgene within the cell. If it is determined that the amount of RNA orprotein expressed from the target gene is lower following contact withthe iRNA agent, then it can be concluded that the iRNA agent downregulates target gene expression. The level of target RNA or protein inthe cell can be determined by any method desired. For example, the levelof target RNA can be determined by Northern blot analysis, reversetranscription coupled with polymerase chain reaction (RT-PCR), or RNAseprotection assay. The level of protein an be determined by, for example,Western blot analysis.

The mRNA agent conjugated with a lipophilic moiety, e.g., a cholesterol,can be tested in an in vitro or/and in an in vivo system. For example,the target gene or a fragment thereof can be fused to a reporter gene ona plasmid. The plasmid can be transfected into a cell, e.g., a neuralcell, with a candidate iRNA agent. The efficacy of the iRNA agent can beevaluated by monitoring expression of the reporter gene. The reportergene can be monitored in vivo, such as by fluorescence or in situhybridization. Exemplary fluorescent reporter genes include but are notlimited to green fluorescent protein and luciferase. Expression of thereporter gene can also be monitored by Northern blot, RT-PCR,RNAse-protection assay, or Western blot analysis as described above.

Efficacy of an iRNA agent conjugated to a lipophilic moiety, e.g.,cholesterol, can be tested in a mammalian cell line, e.g., a mammalianneural cell line, such as a human neuroblastoma cell line. For example,a cell line useful for testing efficacy of an anti-htt iRNA agent is astriatal cell line, e.g. ST14A cell line. Other mammalian neural celllines that can take up an iRNA agent conjugated with a lipophilic moietyinclude, e.g., neuronally differentiated phaeochromocytomas (e.g., PC12cells), primary neuronal cultures (e.g., isolated from a mouse or ratand cultured immediately), and neuroblastoma cell lines. Neuroblastomacell lines include BE(2)-M17, SH-SY5Y (both human) and N2a (mouse).

Controls include:

-   -   (1) testing the efficacy and specificity of an iRNA agent by        assaying for a decrease in expression of the target gene by, for        example, comparison to expression of an endogenous or exogenous        off-target RNA or protein; and    -   (2) testing specificity of the effect on target gene expression        by administering a “nonfunctional” iRNA agent.

Nonfunctional control iRNA agents can:

-   -   (a) target a gene not expressed in the cell;    -   (b) be of nonsensical sequence (e.g., a scrambled version of the        test iRNA); or    -   (c) have a sequence complementary to the target gene, but be        known by previous experiments to lack an ability to silence gene        expression.

A candidate iRNA agent conjugated to a lipophilic molecule can includeother modifications for nuclease resistance. Resistance to a degradentcan be evaluated as follows. A candidate modified iRNA agent (andpreferably a control molecule, usually one that does not include themodification believed to be required for nuclease resistance) can beexposed to degradative conditions, e.g., exposed to a milieu, whichincludes a degradative agent, e.g., a nuclease. E.g., one can use abiological sample, e.g., one that is similar to a milieu, which might beencountered, in therapeutic use, e.g., blood or a cellular fraction,e.g., a cell-free homogenate or disrupted cells. The candidate andcontrol could then be evaluated for resistance to degradation by any ofa number of approaches. For example, the candidate and control could belabeled, preferably prior to exposure, with, e.g., a radioactive orenzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control andmodified RNA's can be incubated with the degradative agent, andoptionally a control, e.g., an inactivated, e.g., heat inactivated,degradative agent. A physical parameter, e.g., size, of the modified andcontrol molecules are then determined. They can be determined by aphysical method, e.g., by polyacrylamide gel electrophoresis or a sizingcolumn, to assess whether the molecule has maintained its originallength, or assessed functionally. Alternatively, Northern blot analysiscan be used to assay the length of an unlabeled modified molecule.

A functional assay can also be used to evaluate the candidate agent. Afunctional assay can be applied initially or after an earliernon-functional assay, (e.g., assay for resistance to degradation) todetermine if the modification alters the ability of the molecule tosilence gene expression. For example, a cell, e.g., a mammalian cell,such as a mouse or human cell, can be co-transfected with a plasmidexpressing a fluorescent protein, e.g., GFP, and a candidate RNA agenthomologous to the transcript encoding the fluorescent protein. Forexample, a modified siRNA homologous to the GFP mRNA can be assayed forthe ability to inhibit GFP expression by monitoring for a decrease incell fluorescence, as compared to a control cell, in which thetransfection did not include the candidate siRNA, e.g., controls with noagent added and/or controls with a non-modified RNA added. Efficacy ofthe candidate agent on gene expression can be assessed by comparing cellfluorescence in the presence of the modified and unmodified iRNA agents.

The effect of the modified iRNA agent on target RNA levels can beverified by Northern blot to assay for a decrease in the level of targetmRNA, or by Western blot to assay for a decrease in the level of targetprotein, as compared to a negative control. Controls can include cellsin which no agent is added and/or cells in which a non-modified iRNAagent is added.

Assays can include time course experiments to monitor stability andduration of silencing by an iRNA agent and monitoring in dividing versusnondividing cells. Presumably in dividing cells, the iRNA agent isdiluted out over time, thus decreasing the duration of the silencingeffect. The implication is that dosage will have to be adjusted in vivo,and/or an iRNA agent will have to be administered more frequently tomaintain the silencing effect. To monitor nondividing cells, cells canbe arrested by serum withdrawal. Neurons are post-mitotic cells, andthus neural cells are aptly suited for assaying the stability of iRNAagents, such as an anti-htt iRNA agent, for use in therapeuticcompositions for the treatment of disorders of the nervous system, e.g.,neurological disorders, such as HD.

A candidate iRNA agent can also be evaluated for cross-speciesreactivity. For example, cell lines derived from different species(e.g., mouse vs. human) or in biological samples (e.g., serum or tissueextracts) isolated from different species can be transfected with acandidate iRNA agent conjugated to a lipophilic moiety, e.g.,cholesterol. The efficacy of the iRNA agent can be determined for thecell from the different species.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent conjugated with a lipophilic agent for enhanceduptake into neural cells can be evaluated with respect to itssusceptibility to cleavage by an endonuclease or exonuclease, such aswhen the iRNA agent is introduced into the body of a subject. Methodscan be employed to identify sites that are susceptible to modification,particularly cleavage, e.g., cleavage by a component found in the bodyof a subject. The component (e.g., an exonuclease or endonuclease) canbe specific for a particular area of the body, such as a particulartissue, organ, or bodily fluid (e.g., blood, plasma, or serum). Sites inan iRNA agent that are susceptible to cleavage, either byendonucleolytic or exonucleolytic cleavage, in certain areas of thebody, may be resistant to cleavage in other areas of the body. Anexemplary method includes:

(1) determining the point or points at which a substance present in thebody of a subject, and preferably a component present in a compartmentof the body into which a therapeutic dsRNA is to be introduced (thisincludes compartments into which the therapeutic is directly introduced,e.g., the circulation, as well as in compartments to which thetherapeutic is eventually targeted; in some cases, e.g, the eye or thebrain the two are the same), cleaves a dsRNA, e.g., an iRNA agent, and

(2) identifying one or more points of cleavage, e.g., endonucleolytic,exonucleolytic, or both, in the dsRNA. Optionally, the method furtherincludes providing an RNA modified to inhibit cleavage at such sites.

These steps can be accomplished by using one or more of the followingassays:

-   -   (i) (a) contacting a candidate dsRNA, e.g., an iRNA agent, with        a test agent (e.g., a biological agent),        -   (b) using a size-based assay, e.g., gel electrophoresis to            determine if the iRNA agent is cleaved. In a preferred            embodiment a time course is taken and a number of samples            incubated for different times are applied to the size-based            assay. In preferred embodiments, the candidate dsRNA is not            labeled. The method can be a “stains all” method.    -   (ii) (a) supplying a candidate dsRNA, e.g., an iRNA agent, which        is radiolabeled;        -   (b) contacting the candidate dsRNA with a test agent,        -   (c) using a size-based assay, e.g., gel electrophoresis to            determine if the iRNA agent is cleaved. In a preferred            embodiment a time course is taken where a number of samples            are incubated for different times and applied to the            size-based assay. In preferred embodiments, the            determination is made under conditions that allow            determination of the number of nucleotides present in a            fragment. E.g., an incubated sample is run on a gel having            markers that allow assignment of the length of cleavage            products. The gel can include a standard that is a “ladder”            digestion. Either the sense or antisense strand can be            labeled. Preferably only one strand is labeled in a            particular experiment. The label can be incorporated at the            5′ end, 3′ end, or at an internal position. Length of a            fragment (and thus the point of cleavage) can be determined            from the size of the fragment based on the ladder and            mapping using a site-specific endonuclease such as RNAse T1.    -   (iii) fragments produced by any method, e.g., one of those        above, can be analyzed by mass spectrometry. Following        contacting the iRNA with the test agent, the iRNA can be        purified (e.g., partially purified), such as by        phenol-chloroform extraction followed by precipitation. Liquid        chromatography can then be used to separate the fragments and        mass spectrometry can be used to determine the mass of each        fragment. This allows determination of the mechanism of        cleavage, e.g., if by direct phosphate cleavage, such as be 5′        or 3′ exonuclease cleavage, or mediated by the 2′OH via        formation of a cyclic phosphate.

More than one iRNA agent, e.g., anti-htt iRNA agent, can be evaluated.The evaluation can be used to select a sequence for use in a therapeuticiRNA agent. For example, it allows the selection of a sequence having anoptimal (usually minimized) number of sites that are cleaved by asubstance(s), e.g., an enzyme, present in the relevant compartments of asubject's body. Two or more iRNA agent candidates can be evaluated toselect a sequence that is optimized. For example, two or more candidatescan be evaluated and the one with optimum properties, e.g., fewercleavage sites, selected.

The information relating to a site of cleavage can be used to select abackbone atom, a sugar or a base, for modification, e.g., a modificationto decrease cleavage.

Exemplary modifications include modifications that inhibitendonucleolytic degradation, including the modifications describedherein. Particularly favored modifications include: 2′ modification,e.g., provision of a 2′ OMe moiety on a U in a sense or antisensestrand, but especially on a sense strand; modification of the backbone,e.g., with the replacement of an O with an S, in the phosphate backbone,e.g., the provision of a phosphorothioate modification, on the U or theA or both, especially on an antisense strand; replacement of the U witha C5 amino linker; replacement of an A with a G (sequence changes arepreferred to be located on the sense strand and not the antisensestrand); and modification of the at the 2′, 6′, 7′, or 8′ position.Preferred embodiments are those in which one or more of thesemodifications are present on the sense but not the antisense strand, orembodiments where the antisense strand has fewer of such modifications.

Exemplary modifications also include those that inhibit degradation byexonucleases. Examples of modifications that inhibit exonucleolyticdegradation can be found herein. Particularly favored modificationsinclude: 2′ modification, e.g., provision of a 2′ OMe moiety in a 3′overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom ofthe molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′position, as indicated by the context); modification of the backbone,e.g., with the replacement of a P with an S, e.g., the provision of aphosphorothioate modification, or the use of a methylated P in a 3′overhang, e.g., at the 3′terminus; combination of a 2′ modification,e.g., provision of a 2′ OMe moiety and modification of the backbone,e.g., with the replacement of a P with an S, e.g., the provision of aphosphorothioate modification, or the use of a methylated P, in a 3′overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl;modification with an abasic pyrrolidine in a 3′ overhang, e.g., at the3′ terminus; modification with naproxen, ibuprofen, or other moietieswhich inhibit degradation at the 3′ terminus.

These methods can be used to select and or optimize a therapeutic iRNAagent conjugated with a lipophilic moiety, e.g., cholesterol, forenhanced uptake into neural cells.

The method can be used to evaluate a candidate iRNA agent conjugatedwith a lipophilic moiety and that also includes a modification that, forexample, inhibits degradation, targets the dsRNA molecule, or modulateshybridization. Such modifications are described herein. A cleavage assaycan be combined with an assay to determine the ability of a modified ornon-modified candidate to silence the target. E.g., one might(optionally) test a candidate to evaluate its ability to silence atarget (or off-target sequence), evaluate its susceptibility tocleavage, modify it (e.g., as described herein, e.g., to inhibitdegradation) to produce a modified candidate, and test the modifiedcandidate for one or both of the ability to silence and the ability toresist degradation. The procedure can be repeated. Modifications can beintroduced one at a time or in groups. A cell-based method can be usedto monitor the ability of the iRNA agent to silence. This can befollowed by a different method, e.g, a whole animal method, to confirmactivity.

A test agent refers to a biological agent, e.g., biological sample,tissue extract or prep, serum, a known enzyme or other molecule known tomodify, e.g., cleave, a dsRNA, e.g., an endonuclease. The test agent canbe in a compartment of the body in which the RNAi agent will be exposed.For example, for an iRNA agent that is administered directly in toneural tissue (e.g., into the brain or into the spinal cord) the testagent could be brain tissue extract or spinal fluid. An iRNA agent thatis to be supplied directly to the eye can be incubated with an extractof the eye.

In Vivo Testing

An iRNA agent conjugated with a lipophilic moiety and identified ashaving an enhanced capability of entering neural cells in culture can betested for functionality in vivo in an animal model (e.g., in a mammal,such as in mouse or rat). For example, the iRNA agent can beadministered to an animal, and the iRNA agent evaluated with respect toits biodistribution, e.g., is uptake into neural cells, its stability,and its ability to inhibit expression of a target gene in the neuralcells.

The iRNA agent can be administered to the animal model in the samemanner that it would be administered to a human. For example, the iRNAagent can be injected directly into a target region of the brain (e.g.,into the cortex, the substantia nigra, the globus pallidus, thehippocampus, or the striatum), and after a period of time, the brain canbe harvested and tissue slices examined for distribution of the agent.

The iRNA agent can also be evaluated for its intracellular distribution.The evaluation can include determining whether the iRNA agent was takenup into the cell. The evaluation can also include determining thestability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNAagent in vivo can be facilitated by use of an iRNA agent conjugated to atraceable marker (e.g., a fluorescent marker such as Cy3, Cy5, FITC,rhodamine, or fluorescein; a radioactive label, such as ³²P, ³³P, or ³H;gold particles; or antigen particles for immunohistochemistry).

An iRNA agent useful for monitoring biodistribution can lack genesilencing activity in vivo. For example, the iRNA agent can target agene not present in the animal (e.g., an iRNA agent injected into mousecan target luciferase), or an iRNA agent can have a non-sense sequence,which does not target any gene, e.g., any endogenous gene).Localization/biodistribution of the iRNA can be monitored by a traceablelabel attached to the iRNA agent, such as a traceable agent describedabove

The iRNA agent conjugated to a lipophilic moiety can be evaluated withrespect to its ability to down regulate target gene expression. Levelsof target gene expression in vivo can be measured, for example, by insitu hybridization, or by the isolation of RNA from tissue prior to andfollowing exposure to the iRNA agent. Target RNA can be detected by anydesired method, including but not limited to RT-PCR, Northern blot, orRNAase protection assay. Alternatively, or additionally, target geneexpression can be monitored by performing Western blot analysis ontissue extracts treated with an iRNA agent.

An iRNA agent conjugated to a lipophilic agent for enhanced uptake intoneural cells can be tested in a mouse model for a neurological disease.For example, an iRNA agent conjugated to a lipophilic agent can betested in a mouse model for HD, such as a mouse carrying a wildtype copyof the human htt gene or in mouse carrying a mutant human htt, e.g., anhtt gene carrying an expanded CAG repeat. A treated mouse model can beobserved for a decrease in symptoms associated with HD, e.g., a decreasein clasping. The treated mouse can be assessed for other phentypes,e.g., expression of DARPP protein in brain cells, such as in mediumspiny neurons of the brain. In one embodiment, such a secondary assayrequires dissection of the mouse brain for Western blot analysis or insitu hybridization of brain tissue, including spiny neurons.

iRNA Chemistry

Described herein are isolated iRNA agents, e.g., dsRNA molecules, thatmediate RNAi. The iRNA agents are modified for enhanced uptake intoneural cells by their attachment to at least one lipophilic moiety,e.g., a cholesterol molecule.

Generally, the iRNA agents featured in the invention should include aregion of sufficient homology to a target gene, e.g., a target geneexpressed in a neural cell, and be of sufficient length in terms ofnucleotides, such that the iRNA agent, or a fragment thereof, canmediate down regulation of the target gene. It is not necessary thatthere be perfect complementarity between the iRNA agent and the target,but the correspondence must be sufficient to enable the iRNA agent, or acleavage product thereof, to direct sequence specific silencing, e.g.,by RNAi cleavage of the target RNA, e.g., mRNA. Therefore, the iRNAagents featured in the instant invention include agents comprising asense strand and antisense strand each comprising a sequence of at least16, 17 or 18 nucleotides which is essentially identical, as definedbelow, to a sequence of a gene expressed in a neural cell, except thatnot more than 1, 2 or 3 nucleotides per strand, respectively, have beensubstituted by other nucleotides (e.g., adenosine replaced by uracil),while essentially retaining the ability to inhibit expression of thetarget gene in a mammalian cell. Exemplary iRNA agents may thereforepossess at least 15 nucleotides identical to the target gene sequence,but include 1, 2 or 3 base mismatches with respect to either the targetmRNA sequence or between the sense and antisense strand are introduced.Mismatches to the target mRNA sequenceparticularly in the antisensestrand, are most tolerated in the terminal regions and if present arepreferably in a terminal region or regions, e.g., within 6, 5, 4, or 3nucleotides of the 5′ and/or 3′ terminus, most preferably within 6, 5,4, or 3 nucleotides of the 5′-terminus of the sense strand or the3′-terminus of the antisense strand. The sense strand need only besufficiently complementary with the antisense strand to maintain theover all double strand character of the molecule.

Single stranded regions of an iRNA agent will often be modified orinclude nucleoside surrogates, e.g., the unpaired region or regions of ahairpin structure, e.g., a region which links two complementary regions,can have modifications or nucleoside surrogates. Modifications tostabilize one or both of the 3′- or 5′-terminus of an iRNA agent, e.g.,against exonucleases, or to favor the antisense sRNA agent to enter intoRISC are also favored. Modifications can include C3 (or C6, C7, C12)amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers(C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol),special biotin or fluorescein reagents that come as phosphoramidites andthat have another DMT-protected hydroxyl group, allowing multiplecouplings during RNA synthesis. As discussed elsewhere herein, an iRNAagent will often be modified or include an SRMS in addition to thenucleotide surrogate. An SRMS replaces a ribose sugar on aribonucleotide with another moiety, e.g., a non-carbohydrate (preferablycyclic) carrier. SRMS' are described in greater detail below.

Although, in mammalian cells, long ds iRNA agents can induce theinterferon response which is frequently deleterious, short ds iRNAagents do not trigger the interferon response, at least not to an extentthat is deleterious to the cell and host. The iRNA agents of the presentinvention include molecules which are sufficiently short that they donot trigger the interferon response in mammalian cells. Thus, theadministration of a composition of an iRNA agent (e.g., formulated asdescribed herein) to a mammalian cell can be used to silence expressionof a gene expressed in a neural cell, e.g., the htt gene, whilecircumventing the interferon response. Molecules that are short enoughthat they do not trigger an interferon response are termed sRNA agentsor shorter iRNA agents herein. “sRNA agent or shorter iRNA agent” asused herein, refers to an iRNA agent, e.g., a double stranded RNA agentor single strand agent, that is sufficiently short that it does notinduce a deleterious interferon response in a human cell, e.g., it has aduplexed region of less than 60 but preferably less than 50, 40, or 30nucleotide pairs.

In addition to homology to target RNA and the ability to down regulate atarget gene, an iRNA agent will preferably have one or more of thefollowing properties:

-   -   (1) it will be of the Formula 1, 2, 3, or 4 described below;    -   (2) if single stranded it will have a 5′ modification that        includes one or more phosphate groups or one or more analogs of        a phosphate group;    -   (3) it will, despite modifications, even to a very large number        of bases, specifically base pair and form a duplex structure        with a homologous target RNA of sufficient thermodynamic        stability to allow modulation of the activity of the targeted        RNA;    -   (4) it will, despite modifications, even to a very large number,        or all of the nucleosides, still have “RNA-like” properties,        i.e., it will possess the overall structural, chemical and        physical properties of an RNA molecule, even though not        exclusively, or even partly, of ribonucleotide-based content.        For example, all of the nucleotide sugars can contain e.g.,        2′OMe, or 2′ fluoro in place of 2′ hydroxyl. This        deoxyribonucleotide-containing agent can still be expected to        exhibit RNA-like properties. While not wishing to be bound by        theory, the electronegative fluorine prefers an axial        orientation when attached to the C2′ position of ribose. This        spatial preference of fluorine can, in turn, force the sugars to        adopt a C_(3′)-endo pucker. This is the same puckering mode as        observed in RNA molecules and gives rise to the        RNA-characteristic A-family-type helix. Further, since fluorine        is a good hydrogen bond acceptor, it can participate in the same        hydrogen bonding interactions with water molecules that are        known to stabilize RNA structures. (Generally, it is preferred        that a modified moiety at the 2′ sugar position will be able to        enter into hydrogen-bonding which is more characteristic of the        2′-OH moiety of a ribonucleotide than the 2′-H moiety of a        deoxyribonucleotide. A preferred iRNA agent will: exhibit a        C_(3′)-endo pucker in all, or at least 50, 75, 80, 85, 90, or        95% of its sugars; exhibit a C_(3′)-endo pucker in a sufficient        amount of its sugars that it can give rise to a the        RNA-characteristic A-family-type helix; will have no more than        20, 10, 5, 4, 3, 2, or 1 sugar which is not a C_(3′)-endo pucker        structure. These limitations are particularly preferably in the        antisense strand;    -   Preferred 2′-modifications with C3′-endo sugar pucker include:

2′-OH, 2′-O-Me, 2′-O-methoxyethyl, 2′-β-aminopropyl,2′-F,2′-O—CH₂—CO—NHMe, 2′-O—CH₂—CH₂-O—CH₂—CH₂-N(Me)₂, LNA

-   -   (5) regardless of the nature of the modification, and even        though the oligonucleotide agent can contain deoxynucleotides or        modified deoxynucleotides, it is preferred that DNA molecules,        or any molecule in which more than 50, 60, or 70% of the        nucleotides in the molecule are deoxyribonucleotides, or        modified deoxyribonucleotides which are deoxy at the 2′        position, are excluded from the definition of oligonucleotide        agent.

A “ds mRNA agent” (abbreviation for “double stranded (ds) iRNA agent”)as used herein, is an iRNA agent which includes more than one, andpreferably two, strands in which interchain hybridization can form aregion of duplex structure.

The antisense strand of a double stranded iRNA agent should be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides inlength. It should be equal to or less than 60, 50, 40, or 30 nucleotidesin length. Preferred ranges are 15 to 30, 17 to 25, 19 to 23, and 19 to21 nucleotides in length.

The sense strand of a double stranded iRNA agent should be equal to orat least 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length.It should be equal to or less than 60, 50, 40, or 30 nucleotides inlength. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21nucleotides in length.

The double strand portion of a double stranded iRNA agent should beequal to or at least, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40,or 50 nucleotide pairs in length. It should be equal to or less than 60,50, 40, or 30 nucleotide pairs in length. Preferred ranges are 15 to 30,17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in length.

It may be desirable to modify one or both of the antisense and sensestrands of a double strand iRNA agent. In some cases they will have thesame modification or the same class of modification but in other casesthe sense and antisense strand will have different modifications, e.g.,in some cases it is desirable to modify only the sense strand. It may bedesirable to modify only the sense strand, e.g., to inactivate it, e.g.,the sense strand can be modified in order to inactivate the sense strandand prevent formation of an active iRNA agent/protein or RISC. This canbe accomplished by a modification which prevents 5′-phosphorylation ofthe sense strand, e.g., by modification with a 5′-O-methylribonucleotide (see Nykanen et al., (2001) ATP requirements and smallinterfering RNA structure in the RNA interference pathway. Cell 107,309-321.) Other modifications which prevent phosphorylation can also beused, e.g., simply substituting the 5′-OH by H rather than O-Me.Alternatively, a large bulky group may be added to the 5′-phosphateturning it into a phosphodiester linkage, though this may be lessdesirable as phosphodiesterases can cleave such a linkage and release afunctional sRNA 5′-end. Antisense strand modifications include 5′phosphorylation as well as any of the other 5′ modifications discussedherein.

It is preferred that the sense and antisense strands be chosen such thatthe ds mRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains sense andantisense strands, preferably paired to contain an overhang, e.g., oneor two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3nucleotides. Most embodiments will have a 3′ overhang. Preferred iRNAagents will have single-stranded overhangs, preferably 3′ overhangs, of1 to 4, or preferably 2 or 3 nucleotides in length at each end. Theoverhangs can be the result of one strand being longer than the other,or the result of two strands of the same length being staggered. 5′ endsare preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe iRNA agent range discussed above. iRNA agents can resemble in lengthand structure the natural Dicer processed products from long dsRNAs.Embodiments in which the two strands of the sRNA agent are linked, e.g.,covalently linked are also included. Hairpin, or other single strandstructures which provide the required double stranded region, andpreferably a 3′ overhang are also within the invention.

As used herein, the phrase “mediates RNAi” refers to the ability of anagent to silence, in a sequence specific manner, a target gene.“Silencing a target gene” means the process whereby a neural cellcontaining and/or secreting a certain product of the target gene whennot in contact with the agent, will contain and/or secret at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product whencontacted with the agent, as compared to a similar neural cell which hasnot been contacted with the agent. Such product of the target gene can,for example, be a messenger RNA (mRNA), a protein, or a regulatoryelement While not wishing to be bound by theory, it is believed thatsilencing by the agents described herein uses the RNAi machinery orprocess and a guide RNA, e.g., an iRNA agent of 15 to 30 nucleotidepairs.

As used herein, the term “complementary” is used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule, e.g., an htt mRNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 4 nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA (e.g., a target htt mRNA) if the iRNAagent reduces the production of a protein encoded by the target mRNA.The iRNA agent may also be “exactly complementary” (excluding the SRMScontaining subunit(s)) to a target RNA, e.g., the target RNA and theiRNA agent can anneal, preferably to form a hybrid made exclusively ofWatson-Crick base pairs in the region of exact complementarity. A“sufficiently complementary” target RNA can include a region (e.g., ofat least 7 nucleotides) that is exactly complementary to a target RNA,e.g., an htt RNA. Moreover, in some embodiments, the iRNA agentspecifically discriminates a single-nucleotide difference.

iRNA agents conjugated with a lipophilic agent for enhanced uptake intoneural cells include iRNA agents which have been further modified, e.g.,to improve efficacy, and polymers of nucleoside surrogates. UnmodifiedRNA refers to a molecule in which the components of the nucleic acid,namely sugars, bases, and phosphate moieties, are the same oressentially the same as that which occur in nature, preferably as occurnaturally in the human body. The art has referred to rare or unusual,but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach etal., Nucleic Acids Res. 22: 2183-2196, 1994. Such rare or unusual RNAs,often termed modified RNAs are typically the result of a posttranscriptional modification and are within the term unmodified RNA, asused herein. Modified RNA, as used herein, refers to a molecule in whichone or more of the components of the nucleic acid, namely sugars, bases,and phosphate moieties, are different from that which occur in nature,preferably different from that which occurs in the human body. Whilethey are referred to as modified RNAs, they will of course, because ofthe modification, include molecules that are not, strictly speaking,RNAs. Nucleoside surrogates are molecules in which the ribophosphatebackbone is replaced with a non-ribophosphate construct that allows thebases to the presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of all of the above are discussed herein.

Much of the discussion below refers to single strand molecules. However,it is understood that a ds iRNA agent, e.g., a partially ds iRNA agent,is required or preferred. Thus, it is understood that double strandedstructures (e.g. where two separate molecules are contacted to form thedouble stranded region or where the double stranded region is formed byintramolecular pairing (e.g., a hairpin structure)) made of the singlestranded structures described below are within the invention. Preferredlengths are described elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or a non-linking 0 of a phosphate moiety. In some cases themodification will occur at all of the subject positions in the nucleicacid but in many, and in fact in most, cases it will not. By way ofexample, a modification may only occur at a 3′ or 5′ terminal position,may only occur in a terminal region, e.g. at a position on a terminalnucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of an RNA or may only occur in a single strand region of an RNA.The ligand can be at attached at the 3′ end, the 5′ end, or at aninternal position, or at a combination of these positions. For example,the ligand can be at the 3′ end and the 5′ end; at the 3′ end and at oneor more internal positions; at the 5′ end and at one or more internalpositions; or at the 3′ end, the 5′ end, and at one or more internalpositions. For example, a phosphorothioate modification at a non-linking0 position may only occur at one or both termini, or may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. Similarly, amodification may occur on the sense strand, antisense strand, or bothstrands. In some cases, the sense and antisense strand will have thesame modifications or the same class of modifications, but in othercases the sense and antisense strand will have different modifications,e.g., in some cases it may be desirable to modify only one strand, e.g.the sense strand. The 5′ end can be phosphorylated. In a particularlypreferred embodiment, the sense strand is modified at the 3′ end by theaddition of a cholesterol.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular bases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of aribonucleic acid. The basic components are the ribose sugar, the base,the terminal phosphates, and phosphate internucleotide linkers. Wherethe bases are naturally occurring bases, e.g., adenine, uracil, guanineor cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (asdepicted) and W, X, Y, and Z are all O, Formula 1 represents a naturallyoccurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in someapplications, e.g., unmodified oligoribonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications to one ormore of the above RNA components can confer improved properties, and,e.g., can render oligoribonucleotides more stable to nucleases.Unmodified oligoribonucleotides may also be less than optimal in termsof offering tethering points for attaching ligands or other moieties toan iRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or moreof:

(i) alteration, e.g., replacement, of one or both of the non-linking (Xand Y) phosphate oxygens and/or of one or more of the linking (W and Z)phosphate oxygens (When the phosphate is in the terminal position, oneof the positions W or Z will not link the phosphate to an additionalelement in a naturally occurring ribonucleic acid. However, forsimplicity of terminology, except where otherwise noted, the W positionat the 5′ end of a nucleic acid and the terminal Z position at the 3′end of a nucleic acid, are within the term “linking phosphate oxygens”as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesalereplacement of the ribose sugar with a structure other than ribose,e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with“dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone(bracket II);

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid but rather modified simply indicates a difference froma naturally occurring molecule.

It is understood that the actual electronic structure of some chemicalentities cannot be adequately represented by only one canonical form(i.e. Lewis structure). While not wishing to be bound by theory, theactual structure can instead be some hybrid or weighted average of twoor more canonical forms, known collectively as resonance forms orstructures. Resonance structures are not discrete chemical entities andexist only on paper. They differ from one another only in the placementor “localization” of the bonding and nonbonding electrons for aparticular chemical entity. It can be possible for one resonancestructure to contribute to a greater extent to the hybrid than theothers. Thus, the written and graphical descriptions of the embodimentsof the present invention are made in terms of what the art recognizes asthe predominant resonance form for a particular species. For example,any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)would be represented by X═O and Y═N in the above figure.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors (cf. Bracket I in Formula 1 above). While not wishing to bebound by theory, it is believed that since the charged phosphodiestergroup is the reaction center in nucleolytic degradation, its replacementwith neutral structural mimics should impart enhanced nucleasestability. Again, while not wishing to be bound by theory, it can bedesirable, in some embodiment, to introduce alterations in which thecharged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates (see Bracket II of Formula 1 above).While not wishing to be bound by theory, it is believed that the absenceof a repetitively charged backbone diminishes binding to proteins thatrecognize polyanions (e.g. nucleases). Again, while not wishing to bebound by theory, it can be desirable in some embodiment, to introducealterations in which the bases are tethered by a neutral surrogatebackbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNAsurrogate.

Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g.,n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotinand fluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance.

Other examples of terminal modifications include dyes, intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Preferred modifications include the additionof a methylphosphonate at the 3′-most terminal linkage; a 3′C5-aminoalkyl-dT; 3′ cationic group; or another 3′ conjugate to inhibit3′-5′ exonucleolytic degradation.

Terminal modifications useful for modulating activity includemodification of the 5′ end with phosphate or phosphate analogs. E.g., inpreferred embodiments iRNA agents, especially antisense strands, are 5′phosphorylated or include a phosphoryl analog at the 5′ terminus.5′-phosphate modifications include those which are compatible with RISCmediated gene silencing. Suitable modifications include:5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP (OH)(O)—O-5′-, (OH)₂(O)P-5′-CH2—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorescein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

Referring to Formula 2 above, R¹, R², and R³ are each, independently, H,(i.e. abasic nucleotides), adenine, guanine, cytosine and uracil,inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine,isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂NHR⁹; NHC(O)R⁸; cyano; mercapto, SR⁸;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ togethercombine with R⁷ to form an [—O—CH₂—] covalently bound bridge between thesugar 2′ and 4′ carbons.

A¹ is:

; H; OH; OCH₃; W¹; an abasic nucleotide; or absent;

(a preferred A1, especially with regard to anti-sense strands, is chosenfrom 5′-monophosphate ((HO)₂(O)P—O-5′), 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′),5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-)).

A² is:

A³ is:

and

A⁴ is:

; H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n) SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰;O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰, NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰;O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰ or —O—. W⁴ is O, CH₂, NH, or S.

X¹, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, O⁻, OR⁸, S, Se, BH₃ ⁻,H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each ofwhich may be optionally substituted.

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH,(CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰,O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰; NH(CH₂)_(n)SR¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰,O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰,O-Q-R¹⁰, S-Q-R¹⁰.

X is 5-100, chosen to comply with a length for an RNA agent describedherein.

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [—O—CH₂—]covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5dyes); sulfur, silicon, boron or ester protecting group; intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (cholesterol, cholicacid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabeledmarkers, enzymes, haptens (e.g. biotin), transport/absorptionfacilitators (e.g., aspirin, vitamin E, folic acid), syntheticribonucleases (e.g., imidazole, bisimidazole, histamine, imidazoleclusters, acridine-imidazole conjugates, Eu3+ complexes oftetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20.Q is a spacer selected from the group consisting of abasic sugar, amide,carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea,sulfonamide, or morpholino, biotin or fluorescein reagents.

Preferred RNA agents in which the entire phosphate group has beenreplaced have the following structure (see Formula 3 below):

Referring to Formula 3, A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent,in which L is a linker, wherein one or both L may be present or absentand is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g);O(CH₂)_(g); S(CH₂)_(g). G is a functional group selected from the groupconsisting of siloxane, carbonate, carboxymethyl, carbamate, amide,thioether, ethylene oxide linker, sulfonate, sulfonamide,thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

R¹⁰, R²⁰, and R³⁰ are each, independently, H, (i.e. abasic nucleotides),adenine, guanine, cytosine and uracil, inosine, thymine, xanthine,hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂R⁹; NHC(O)R⁸; cyano; mercapto, SR⁷;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰together combine with R⁷⁰ to form an [—O—CH₂—] covalently bound bridgebetween the sugar 2′ and 4′ carbons.

x is 5-100 or chosen to comply with a length for an RNA agent describedherein.

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an[—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid. m is 0-1,000,000, n is 0-20, and g is 0-2.

Preferred nucleoside surrogates have the following structure (seeFormula 4 below):

SLR¹⁰⁰-(M-SLR²⁰⁰)_(x)-M-SLR³⁰⁰  FORMULA 4

S is a nucleoside surrogate selected from the group consisting ofmophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is alinker and is selected from the group consisting of CH₂(CH₂)_(g);N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g); —C(O)(CH₂)_(n)— or may be absent. Mis an amide bond; sulfonamide; sulfinate; phosphate group; modifiedphosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasicnucleotides), adenine, guanine, cytosine and uracil, inosine, thymine,xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil substituted 1,2,4,-triazoles, 2-pyridinones,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

x is 5-100, or chosen to comply with a length for an RNA agent describedherein; and g is 0-2.

Enhanced Nuclease Resistance

An iRNA agent conjugated to a lipophilic moiety for enhanced uptake intoneural cells can have enhanced resistance to nucleases.

For increased nuclease resistance and/or binding affinity to the target,an iRNA agent, e.g., the sense and/or antisense strands of the iRNAagent, can include, for example, 2′-modified ribose units and/orphosphorothioate linkages. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE andaminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino). It isnoteworthy that oligonucleotides containing only the methoxyethyl group(MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilitiescomparable to those modified with the robust phosphorothioatemodification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality.

Preferred substitutents are 2′-methoxyethyl, 2′-OCH3,2′-O-allyl,2′-C-allyl, and 2′-fluoro.

In certain aspects, nuclease resistance of iRNA agents is enhanced byidentifying nuclease-susceptible sites and modifying such sites toinhibit cleavage. For example, the dinucleotides 5′-UA-3′, 5′ UG3′,5′-CA-3′, 5′ UU-3′, or 5′-CC-3′ can serve as cleavage sites. Enhancednuclease resistance can therefore be achieved by modifying the 5′nucleotide, resulting, for example, in at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine is a2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; atleast one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide; at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide; or at least one 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide. The iRNA agent can include at least 2, at least 3, at least4 or at least 5 of such dinucleotides. In certain embodiments, all thepyrimidines of an iRNA agent carry a 2′-modification, and the iRNA agenttherefore has enhanced resistance to endonucleases.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The inclusion of furanose sugars in the oligonucleotide backbone canalso decrease endonucleolytic cleavage. An iRNA agent can be furthermodified by including a 3′ cationic group, or by inverting thenucleoside at the 3′-terminus with a 3′-3′ linkage. In anotheralternative, the 3′-terminus can be blocked with an aminoalkyl group,e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′exonucleolytic cleavage. While not being bound by theory, a 3′conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the3′-end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

Similarly, 5′ conjugates can inhibit 5′-3′ exonucleolytic cleavage.While not being bound by theory, a 5′ conjugate, such as naproxen oribuprofen, may inhibit exonucleolytic cleavage by sterically blockingthe exonuclease from binding to the 5′-end of oligonucleotide. Evensmall alkyl chains, aryl groups, or heterocyclic conjugates or modifiedsugars (D-ribose, deoxyribose, glucose etc.) can block3′-5′-exonucleases.

An iRNA agent can have increased resistance to nucleases when a duplexediRNA agent includes a single-stranded nucleotide overhang on at leastone end. In preferred embodiments, the nucleotide overhang includes 1 to4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment,the unpaired nucleotide of the single-stranded overhang that is directlyadjacent to the terminal nucleotide pair contains a purine base, and theterminal nucleotide pair is a G-C pair, or at least two of the last fourcomplementary nucleotide pairs are G-C pairs. In further embodiments,the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in anexemplary embodiment the nucleotide overhang is 5′-GC-3′. In preferredembodiments, the nucleotide overhang is on the 3′-end of the antisensestrand. In one embodiment, the iRNA agent includes the motif 5′-CGC-3′on the 3′-end of the antisense strand, such that a 2-nt overhang5′-GC-3′ is formed.

Thus, an iRNA agent can include modifications so as to inhibitdegradation, e.g., by nucleases, e.g., endonucleases or exonucleases,found in the body of a subject. These monomers are referred to herein asNRMs, or Nuclease Resistance promoting Monomers, the correspondingmodifications as NRM modifications. In many cases these modificationswill modulate other properties of the iRNA agent as well, e.g., theability to interact with a protein, e.g., a transport protein, e.g.,serum albumin, or a member of the RISC, or the ability of the first andsecond sequences to form a duplex with one another or to form a duplexwith another sequence, e.g., a target molecule.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent.

NRM modifications include some which can be placed only at the terminusand others which can go at any position. Generally, these modificationscan inhibit hybridization so it is preferable to use them only interminal regions, and preferable not to use them at the cleavage site orin the cleavage region of a sequence which targets a subject sequence orgene, particularly on the antisense strand. They can be used anywhere ina sense strand, provided that sufficient hybridization between the twostrands of the ds iRNA agent is maintained. In some embodiments it isdesirable to put the NRM at the cleavage site or in the cleavage regionof a sense strand, as it can minimize off-target silencing.

In most cases, the NRM modifications will be distributed differentlydepending on whether they are included on a sense or antisense strand.If on an antisense strand, modifications which interfere with or inhibitendonuclease cleavage should not be inserted in the region which issubject to RISC mediated cleavage, e.g., the cleavage site or thecleavage region. As used herein cleavage site refers to the nucleotideon either side of the cleavage site, on the target or on the iRNA agentstrand which hybridizes to it. Cleavage region means a nucleotide within1, 2, or 3 nucleotides of the cleavage site, in either direction.

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus.

Ribose Mimics

The monomers and methods described herein can be used to prepare anoligonucleotide agent, that incorporates a ribose mimic.

Thus, an aspect of the invention features an iRNA agent that includes asecondary hydroxyl group, which can increase efficacy and/or confernuclease resistance to the agent. Nucleases, e.g., cellular nucleases,can hydrolyze nucleic acid phosphodiester bonds, resulting in partial orcomplete degradation of the nucleic acid. The secondary hydroxy groupconfers nuclease resistance to an iRNA agent by rendering the iRNA agentless prone to nuclease degradation relative to an iRNA which lacks themodification. While not wishing to be bound by theory, it is believedthat the presence of a secondary hydroxyl group on the iRNA agent canact as a structural mimic of a 3′ ribose hydroxyl group, thereby causingit to be less susceptible to degradation.

The secondary hydroxyl group refers to an “OH” radical that is attachedto a carbon atom substituted by two other carbons and a hydrogen. Thesecondary hydroxyl group that confers nuclease resistance as describedabove can be part of any acyclic carbon-containing group. The hydroxylmay also be part of any cyclic carbon-containing group, and preferablyone or more of the following conditions is met (1) there is no ribosemoiety between the hydroxyl group and the terminal phosphate group or(2) the hydroxyl group is not on a sugar moiety which is coupled to abase. The hydroxyl group is located at least two bonds (e.g., at leastthree bonds away, at least four bonds away, at least five bonds away, atleast six bonds away, at least seven bonds away, at least eight bondsaway, at least nine bonds away, at least ten bonds away, etc.) from theterminal phosphate group phosphorus of the iRNA agent. In preferredembodiments, there are five intervening bonds between the terminalphosphate group phosphorus and the secondary hydroxyl group.

Preferred mRNA agent delivery modules with five intervening bondsbetween the terminal phosphate group phosphorus and the secondaryhydroxyl group have the following structure (see formula Y below):

Referring to formula Y, A is an iRNA agent, including any iRNA agentdescribed herein. The iRNA agent may be connected directly or indirectly(e.g., through a spacer or linker) to “W” of the phosphate group. Thesespacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—,—(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6),abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether,disulfide, thiourea, sulfonamide, or morpholino, or biotin andfluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified(e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group,W and Z can be independently NH, O, or S; and X and Y can beindependently S, Se, BH₃ ⁻, C₁-C₆ alkyl, C₆-C₁₀ aryl, H, O, O⁻, alkoxyor amino (including alkylamino, arylamino, etc.). Preferably, W, X and Zare O and Y is S.

R₁ and R₃ are each, independently, hydrogen; or C₁-C₁₀₀ alkyl,optionally substituted with hydroxyl, amino, halo, phosphate or sulfateand/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R₂ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₂ may be taken togetherwith R₄ or R₆ to form a ring of 5-12 atoms.

R₄ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₄ may be taken togetherwith R₂ or R₅ to form a ring of 5-12 atoms.

R₅ is hydrogen, C₁-C₁₀₀ alkyl optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₅ may be taken togetherwith R₄ to form a ring of 5-12 atoms.

R₆ is hydrogen, C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl, or, when n is 1, R₆ may be taken togetherwith R₂ to form a ring of 6-10 atoms;

R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_(q)C(O)NHR₉; T is hydrogenor a functional group; n and q are each independently 1-100; R₈ isC₁-C₁₀ alkyl or C₆-C₁₀ aryl; and R₉ is hydrogen, C1-C10 alkyl, C6-C10aryl or a solid support agent.

Preferred embodiments may include one of more of the following subsetsof iRNA agent delivery modules.

In one subset of RNAi agent delivery modules, A can be connecteddirectly or indirectly through a terminal 3′ or 5′ ribose sugar carbonof the RNA agent.

In another subset of RNAi agent delivery modules, X, W, and Z are 0 andY is S.

In still yet another subset of RNAi agent delivery modules, n is 1, andR₂ and R₆ are taken together to form a ring containing six atoms and R₄and R₅ are taken together to form a ring containing six atoms.Preferably, the ring system is a trans-decalin. For example, the RNAiagent delivery module of this subset can include a compound of Formula(Y-1):

The functional group can be, for example, a targeting group (e.g., asteroid or a carbohydrate), a reporter group (e.g., a fluorophore), or alabel (an isotopically labeled moiety). The targeting group can furtherinclude protein binding agents, endothelial cell targeting groups (e.g.,RGD peptides and mimetics), cancer cell targeting groups (e.g., folateVitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates,polyglutamates, polyaspartates), multivalent mannose (for e.g.,macrophage testing), lactose, galactose, N-acetyl-galactosamine,monoclonal antibodies, glycoproteins, lectins, melanotropin, orthyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the formulae herein will be evident to those ofordinary skill in the art. The synthesized compounds can be separatedfrom a reaction mixture and further purified by a method such as columnchromatography, high pressure liquid chromatography, orrecrystallization. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Sugar Replacement Modification Subunit

An iRNA agent modified for enhanced uptake into a neural cell is coupledto a ligand, e.g., a lipophilic ligand. The ligand can be attached tothe oligonucleotide agent through a monomer, e.g., a chemically modifiedmonomer that is integrated into the oligonucleotide agent.

In a preferred embodiment, the coupling is by a tether or a linker (orboth) as described herein, and the complex has the formula representedby:

Ligand-[linker]_(optional)-[tether]_(optional)-oligonucleotide agent

While, in most cases, embodiments are described with respect to anoligonucleotide agent including a number of nucleotides, the inventionincludes monomeric subunits having the structure:

Ligand-[linker]_(optional)-[tether]_(optional)-monomer

Methods of making and incorporating the monomers into theoligonucleotide agents and methods of using of those agents are includedin the invention.

In preferred embodiments, the sugar, e.g., the ribose sugar of one ormore of the nucleotides, (e.g., ribonucleotide, deoxynucleotide, ormodified nucleotide) subunits of an oligonucleotide agent can bereplaced with another moiety, e.g., a non-carbohydrate (preferablycyclic) carrier. A nucleotide subunit in which the sugar of the subunithas been so replaced is referred to herein as a sugar replacementmodification subunit (SRMS). This is often referred to herein as a“tether.” A cyclic carrier may be a carbocyclic ring system, i.e., allring atoms are carbon atoms or a heterocyclic ring system, i.e., one ormore ring atoms may be a heteroatom, e.g., nitrogen, oxygen, or sulfur.The cyclic carrier may be a monocyclic ring system, or may contain twoor more rings, e.g. fused rings. The cyclic carrier may be a fullysaturated ring system, or it may contain one or more double bonds.

The carriers further include (i) at least two “backbone attachmentpoints” and (ii) at least one “tethering attachment point.” A “backboneattachment point” as used herein refers to a functional group, e.g. ahydroxyl group, or generally, a bond available for, and that is suitablefor incorporation of the carrier into the backbone, e.g., the phosphate,or modified phosphate, e.g., sulfur containing, backbone, of aribonucleic acid. A “tethering attachment point” as used herein refersto a constituent ring atom of the cyclic carrier, e.g., a carbon atom ora heteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., aligand, e.g., a targeting or delivery moiety, or a moiety which alters aphysical property. One of the most preferred moieties is a moiety whichpromotes entry into a cell, e.g., a lipophilic moiety, e.g.,cholesterol. While not wishing to be bound by theory it is believed theattachment of a lipophilic agent increases the lipophilicity of anoligonucleotide agent. Optionally, the selected moiety is connected byan intervening tether to the cyclic carrier. Thus, it will often includea functional group, e.g., an amino group, or generally, provide a bond,that is suitable for incorporation or tethering of another chemicalentity, e.g., a ligand to the constituent ring.

Incorporation of one or more SRMSs described herein into anoligonucleotide agent, particularly when tethered to an appropriateentity, can confer one or more new properties to the oligonucleotideagent and/or alter, enhance or modulate one or more existing propertiesin the oligonucleotide agent. E.g., it can alter one or more oflipophilicity or nuclease resistance. Incorporation of one or more SRMSsdescribed herein into an oligonucleotide agent can, particularly whenthe SRMS is tethered to an appropriate entity, modulate, e.g., increase,binding affinity of an oligonucleotide agent to a target RNA, e.g., apre-mRNA, mRNA, or miRNA of the subject or a pathogen of the subject.Incorporation of one or more SRMSs can alter distribution, target theoligonucleotide agent to a particular part of the body, modify theinteraction with nucleic acid binding proteins (e.g., during RISCformation and strand separation), or increase sequence specificity, e.g,to inhibit off-site targeting.

Accordingly, in one aspect, the invention features, an oligonucleotideagent preferably comprising at least one subunit having the structure offormula (I):

wherein:

X is N(CO)R⁷, NR⁷ or CH₂;

Y is NR⁸, O, S, CR⁹R¹⁰, or absent;

Z is CR¹¹R¹² or absent;

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a),OR^(b), (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b), provided that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^(a) or OR^(b) and that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b)(when the SRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ willinclude R^(a) and one will include R^(b); when the SRMSS is internal,two of R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R^(b)); furtherprovided that preferably OR^(a) may only be present with (CH₂)_(n)OR^(b)and (CH₂)_(n)OR^(a) may only be present with OR^(b);

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, C₁-C₆ alkyloptionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ togetherare C₃-C₈ cycloalkyl optionally substituted with R¹⁴;

R⁷ can be a ligand, e.g., R⁷ can be R^(d), or R⁷ can be a ligandtethered indirectly to the carrier, e.g., through a tethering moiety,e.g., C₁-C₂₀ alkyl substituted with NR^(c)R^(d); or C₁-C₂₀ alkylsubstituted with NHC(O)R^(d);

R⁸ is C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR^(c)R⁷;

R^(a) is:

R^(b) is:

Each of A and C is, independently, O or S;

B is OH, O⁻, or

R^(c) is H or C₁-C₆ alkyl;

R^(d) is H or a ligand, e.g., a lipophilic ligand, e.g., cholesterol;and

n is 1-4.

Embodiments can include one or more of the following features:

R¹ can be CH₂OR^(a) and R³ can be OR^(b); or R¹ can be CH₂OR^(a) and R⁹can be OR^(b); or R¹ can be CH₂OR^(a) and R² can be OR^(b).

R¹ can be CH₂OR^(b) and R³ can be OR^(b); or R¹ can be CH₂OR^(b) and R⁹can be OR^(b); or R¹ can be CH₂OR^(b) and R² can be OR^(b); or R¹ can beCH₂OR^(b) and R³ can be OR^(a); or R¹ can be CH₂OR^(b) and R⁹ can beOR^(a); or R¹ can be CH₂OR^(b) and R² can be OR^(a).

R¹ can be OR^(a) and R³ can be CH₂OR^(b); or R¹ can be OR^(a) and R⁹ canbe CH₂OR^(b); or R¹ can be OR^(a) and R² can be CH₂OR^(b).

R¹ can be OR^(b) and R³ can be CH₂OR^(b); or R¹ can be OR^(b) and R⁹ canbe CH₂OR^(b); or R¹ can be OR^(b) and R² can be CH₂OR^(b); or R¹ can beOR^(b) and R³ can be CH₂OR^(a); or R¹ can be OR^(b) and R⁹ can beCH₂OR^(a); or R¹ can be OR^(b) and R² can be CH₂OR^(a).

R³ can be CH₂OR^(a) and R⁹ can be OR^(b); or R³ can be CH₂OR^(a) and R⁴can be OR^(b).

R³ can be CH₂OR^(b) and R⁹ can be OR^(b); or R³ can be CH₂OR^(b) and R⁴can be OR^(b); or R³ can be CH₂OR^(b) and R⁹ can be OR^(a); or R³ can beCH₂OR^(b) and R⁴ can be OR^(a).

R³ can be OR^(b) and R⁹ can be CH₂OR^(a); or R³ can be OR^(b) and R⁴ canbe CH₂OR^(a); or R³ can be OR^(b) and R⁹ can be CH₂OR^(b); or R³ can beOR^(b) and R⁴ can be CH₂OR^(b).

R³ can be OR^(a) and R⁹ can be CH₂OR^(b); or R³ can be OR^(a) and R⁴ canbe CH₂OR^(b).

R⁹ can be CH₂OR^(a) and R¹⁰ can be OR^(b).

R⁹ can be CH₂OR^(b) and R¹⁰ can be OR^(b); or R⁹ can be CH₂OR^(b) andR¹⁰ can be OR^(a).

In a preferred embodiment the ribose is replaced with a pyrrolinescaffold or with a 4-hydroxyproline-derived scaffold, and X is N(CO)R⁷or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

R¹ and R³ can be cis or R¹ and R³ can be trans.

n can be 1.

A can be O or S.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); or R¹ can be(CH₂)_(n)OR^(a) and R³ can be OR^(b).

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be chosen from afolic acid radical; a cholesterol radical; a carbohydrate radical; avitamin A radical; a vitamin E radical; a vitamin K radical. Preferably,R^(d) is a cholesterol radical.

R¹ can be OR^(b) and R³ can be (CH₂)_(n)OR^(b); or R¹ can be OR^(b) andR³ can be (CH₂)_(n)OR^(a); or

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b); or R¹ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(a).

R¹ and R⁹ can be cis or R¹ and R⁹ can be trans.

R¹ can be OR^(a) and R⁹ can be(CH₂)_(n)OR^(b); or R¹ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or R¹ can be (CH₂)_(n)OR^(a) andR⁹ can be OR^(b); or R¹ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(b); orR¹ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(a).

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(a); or R³ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or

R³ can be (CH₂)_(n)OR^(a) and R⁹ can be OR^(b); or R³ can be OR^(a) andR⁹ can be (CH₂)_(n)OR^(b); R³ can be OR^(b) and R⁹ can be(CH₂)_(n)OR^(b); or R³ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(a).

R³ and R⁹ can be cis or R³ and R⁹ can be trans.

In other preferred embodiments the ribose is replaced with a piperidinescaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².

R⁹ can be (CH₂)_(n)OR^(b) and R¹⁰ can be OR^(a).

n can be 1 or 2.

R⁹ can be (CH₂)₁₀R^(b) and R¹⁰ can be OR^(b); or R⁹ can be (CH₂)₁₀R^(a)and R¹⁰ can be OR^(b).

A can be O or S.

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be selected from afolic acid radical; a cholesterol radical; a carbohydrate radical; avitamin A radical; a vitamin E radical; a vitamin K radical. Preferably,R^(d) is a cholesterol radical.

R³ can be (CH₂)₁₀R^(b) and R⁴ can be OR^(a); or R³ can be(CH₂)_(n)OR^(b) and R⁴ can be OR^(b); or

R³ can be (CH₂)_(n)OR^(a) and R⁴ can be OR^(b).

R¹ can be (CH₂)₁₀R^(b) and R² can be OR^(a); or R¹ can be(CH₂)_(n)OR^(b) and R² can be OR^(b); or R¹ can be (CH₂)_(n)OR^(a) andR² can be OR^(b).

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(a).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or R³ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(a); or R³ can be (CH₂)_(n)OR^(a) andR⁹ can be OR^(b).

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis, or R¹ and R³ can be trans.

R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b).

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a piperazinescaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹².

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis or R¹ and R³ can be trans.

n can be 1.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); or R¹ can be(CH₂)_(n)OR^(a) and R³ can be OR^(b).

A can be O or S, preferably S.

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be chosen from thegroup of a folic acid radical; a cholesterol radical; a carbohydrateradical; a vitamin A radical; a vitamin E radical; a vitamin K radical.Preferably, R^(d) is a cholesterol radical.

R⁸ can be CH₃.

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a morpholinoscaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹²

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis, or R¹ and R³ can be trans.

n can be 1.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); of R¹ can be(CH₂)_(n)OR^(a) and R³ can be OR^(b).

Acan be O or S.

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be chosen from thegroup of a folic acid radical; a cholesterol radical; a carbohydrateradical; a vitamin A radical; a vitamin E radical; a vitamin K radical.Preferably, R^(d) is a cholesterol radical.

R⁸ can be CH₃.

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a decalinscaffold, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹together are C⁶ cycloalkyl.

R⁶ can be C(O)NHR⁷.

R¹² can be hydrogen.

R⁶ and R¹² can be trans.

R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

n can be 1 or 2.

R³ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(b); or R³ can be OR^(b) andR⁹ can be (CH₂)_(n)OR^(a).

A can be O or S.

R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) can be chosen from thegroup of a folic acid radical; a cholesterol radical; a carbohydrateradical; a vitamin A radical; a vitamin E radical; a vitamin K radical.Preferably, R^(d) is a cholesterol radical.

In other preferred embodiments the ribose is replaced with adecalin/indane scaffold, e.g., X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²;and R⁵ and R¹¹ together are C⁵ cycloalkyl.

R⁶ can be CH₃.

R¹² can be hydrogen.

R⁶ and R¹² can be trans.

R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

n can be 1 or 2.

R³ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(a); or R³ can be OR^(b) andR⁹ can be (CH₂)_(n)OR^(a).

A can be 0 or S.

R¹⁴ can be N(CH₃)R⁷. R⁷ can be (CH₂)₅NHR^(d) or (CH₂)₅NHR^(d). R^(d) canbe chosen from the group of a folic acid radical; a cholesterol radical;a carbohydrate radical; a vitamin A radical; a vitamin E radical; avitamin K radical. Preferably, R^(d) is a cholesterol radical.

In another aspect, this invention features an oligonucleotide agentcomprising at least one subunit having a structure of formula (II):

X is N(CO)R⁷ or NR⁷;

Each of R¹ and R² is, independently, OR^(a), OR^(b), (CH₂)_(n)OR^(a), or(CH₂)_(n)OR^(b), provided that one of R¹ and R² is OR^(a) or OR^(b) andthe other is (CH₂)_(n)OR^(a) or (CH₂)_(n)OR^(b) (when the SRMS isterminal, one of R¹ or R² will include R^(a) and one will include R^(b);when the SRMSS is internal, both R¹ and R² will each include an R^(b));further provided that preferably OR^(a) may only be present with(CH₂)_(n)OR^(b) and (CH₂)_(n)OR^(a) may only be present with OR^(b);

R⁷ is C₁-C₂₀ alkyl substituted with NR^(c)R^(d);

R⁸ is C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR^(c)R⁷;

R^(a) is:

R^(b) is

Each of A and C is, independently, O or S;

B is OH, O⁻, or

R^(c) is H or C₁-C₆ alkyl;

R^(d) is H or a ligand; and

n is 1-4.

The oligonucleotide agent of the conjugate is substantiallysingle-stranded and comprises from about 12 to about 29 subunits,preferably about 15 to about 25 subunits. An oligonucleotide agent thatis substantially single-stranded includes at least 60%, 70%, 80%, or 90%or more nucleotides that are not duplexed.

Embodiments can include one or more of the features described above.

In a further aspect, this invention features an oligonucleotide agenthaving at least one subunit comprising formula (I) or formula (II).

In one aspect, this invention features an oligonucleotide agent havingat least two subunits comprising formula (I) and/or formula (II).

In another aspect, this invention provides a method of making anoligonucleotide agent described herein having at least one subunitcomprising formula (I) and/or (II). In a further aspect, this inventionprovides a method of modulating expression of a target gene. The methodincludes administering an oligonucleotide agent described herein havingat least one subunit comprising formula (I) and/or (II) to a subject.

In one aspect, this invention features a pharmaceutical compositionhaving an oligonucleotide agent described herein having at least onesubunit comprising formula (I) and/or (II) and a pharmaceuticallyacceptable carrier.

SRMSs or tethers described herein may be incorporated into anyoligonucleotide agent described herein. An oligonucleotide agent mayinclude one or more of the SRMSs described herein. An SRMS can beintroduced at one or more points in an oligonucleotide agent. An SRMScan be placed at or near (within 1, 2, or 3 positions) the 3′ or 5′ endof the oligonucleotide. In some embodiments, it is preferred to not havean SRMS at or near (within 1, 2, or 3 positions of) the 5′ end of theoligonucleotide. An SRMS can be internal, and will preferably bepositioned in regions not critical for binding to the target.

In an embodiment, an oligonucleotide agent may have an SRMS at (orwithin 1, 2, or 3 positions of) the 3′ end.

In another embodiment, an oligonucleotide agent may have an SRMS at aninternal position. In other embodiments, an oligonucleotide agent mayhave an SRMS at the 3′ end and an SRMS at an internal position.

Other modifications to sugars, bases, or backbones described herein canbe incorporated into the oligonucleotide agents.

The oligonucleotide agents can take an architecture or structuredescribed herein.

The oligonucleotide agent can be selected to target any of a broadspectrum of genes, including any of the genes described herein.

In a preferred embodiment the oligonucleotide agent has an architecture(architecture refers to one or more of the overall length) describedherein. In addition to the SRMS-containing bases of the oligonucleotideagents described herein can include nuclease resistant monomers (NRMs).

In another aspect, the invention features an oligonucleotide agent towhich is conjugated a lipophilic moiety, e.g., cholesterol, e.g., byconjugation to an SRMS of an oligonucleotide agent. In a preferredembodiment, the lipophilic moiety enhances entry of the oligonucleotideagent into a cell. In a preferred embodiment, the cell is part of anorganism, tissue, or cell line, e.g., a primary cell line, immortalizedcell line, or any type of cell line disclosed herein. Thus, theconjugated oligonucleotide agent can be used to inhibit expression of atarget gene in an organism, e.g., a mammal, e.g., a human, or to inhibitexpression of a target gene in a cell line or in cells which are outsidean organism.

The lipophilic moiety can be chosen, for example, from the groupconsisting of a lipid, cholesterol, oleyl, retinyl, cholesterylresidues, cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. A preferredlipophilic moiety is cholesterol.

The oligonucleotide agent can have at least one subunit having formula(I) or formula (II) incorporated into it. The oligonucleotide agent canhave one or more of any of the features described herein. For example,when the subunit is of formula (I), R^(d) can be cholesterol; X can beN(CO)R⁷ or NR⁷, Y can be CR⁹R¹⁰, and Z can be absent, and R¹ can be(CH₂)_(n)OR^(b) and R³ can be OR^(a); X can be N(CO)R⁷ or NR⁷, Y can beCR⁹R¹⁰, and Z can be CR¹¹R¹², and R⁹ can be (CH₂)_(n)OR^(b) and R¹⁰ canbe OR^(a); X can be N(CO)R⁷ or NR⁷, Y can be NR⁸, and Z can be CR¹¹R¹²and R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a); X can be CH₂; Y canbe CR⁹R¹⁰; and Z can be CR¹¹R¹², in which R⁶ can be C(O)NHR⁷; or X canbe CH₂; Y can be CR⁹R¹⁰; and Z can be CR¹¹R¹², in which R¹¹ or R¹² canbe C(O)NHR⁷ or R⁵ and R¹¹ together can be C₅ or C₆ cycloalkylsubstituted with N(CH3)R⁷.

Tethered Ligands

A wide variety of entities can be tethered to an oligonucleotide agent,e.g., to the carrier of a ligand-conjugated monomer. Examples aredescribed below in the context of a ligand-conjugated monomer but thatis only one preferred embodiment. Entities can be coupled at otherpoints to an oligonucleotide agent.

A ligand tethered to an oligonucleotide agent (e.g., an oligonucleotideagent targeting an miRNA) can have a favorable effect on the agent. Forexample, the ligand can improve stability, hybridization thermodynamicswith a target nucleic acid, targeting to a particular tissue orcell-type, or cell permeability, e.g., by an endocytosis-dependent or-independent mechanism. Ligands and associated modifications can alsoincrease sequence specificity and consequently decrease off-sitetargeting.

A tethered ligand can include one or more modified bases or sugars thatcan function as intercalators. These are preferably located in aninternal region, such as in a bulge of a miRNA/target duplex. Theintercalator can be an aromatic, e.g., a polycyclic aromatic orheterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systems with 2, 3, or 4 fusedrings. The universal bases described herein can be included on a ligand.

In one embodiment, the ligand can include a cleaving group thatcontributes to target gene inhibition by cleavage of the target nucleicacid. The cleaving group can be, for example, a bleomycin (e.g.,bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline(e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lystripeptide), or metal ion chelating group. The metal ion chelating groupcan include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, oracridine, which can promote the selective cleavage of target RNA at thesite of the bulge by free metal ions, such as Lu(III). In someembodiments, a peptide ligand can be tethered to a miRNA to promotecleavage of the target RNA, e.g., at the bulge region. For example,1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can beconjugated to a peptide (e.g., by an amino acid derivative) to promotetarget RNA cleavage.

A tethered ligand can be an aminoglycoside ligand, which can cause anoligonucleotide agent to have improved hybridization properties orimproved sequence specificity. Exemplary aminoglycosides includeglycosylated polylysine, galactosylated polylysine, neomycin B,tobramycin, kanamycin A, and acridine conjugates of aminoglycosides,such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine,Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog canincrease sequence specificity. For example, neomycin B has a highaffinity for RNA as compared to DNA, but low sequence-specificity. Anacridine analog, neo-5-acridine has an increased affinity for the HIVRev-response element (RRE). In some embodiments the guanidine analog(the guanidinoglycoside) of an aminoglycoside ligand is tethered to anoligonucleotide agent. In a guanidinoglycoside, the amine group on theamino acid is exchanged for a guanidine group. Attachment of a guanidineanalog can enhance cell permeability of an oligonucleotide agent, e.g.,an oligonucleotide agent targeting an miRNA or pre-miRNA.

A tethered ligand can be a poly-arginine peptide, peptoid orpeptidomimetic, which can enhance the cellular uptake of anoligonucleotide agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe ligand-conjugated carrier. In preferred embodiments, the ligand isattached to the carrier via an intervening tether. As discussed above,the ligand or tethered ligand may be present on the monomer when themonomer is incorporated into the growing strand. In some embodiments,the ligand may be incorporated into a “precursor” a ligand-conjugatedmonomer subunit after a “precursor” a ligand-conjugated monomer has beenincorporated into the growing strand. For example, a monomer having,e.g., an amino-terminated tether, e.g., TAP-(CH₂)_(n)NH₂ may beincorporated into a growing oligonucleotide strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer into thestrand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor monomer subunit by coupling the electrophilic group ofthe ligand with the terminal nucleophilic group of the precursor monomersubunit tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an oligonucleotide agent into which it is incorporated. Inpreferred embodiments a ligand provides an enhanced affinity for aselected target, e.g, molecule, cell or cell type, compartment, e.g., acellular or organ compartment, tissue, organ or region of the body, as,e.g., compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin,diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin,Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g.,folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins,protein binding agents, integrin targeting molecules, polycationics,peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine, multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines and substituted acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine,phenanthroline, pyrenes), lys-tyr-lys tripeptide, aminoglycosides,guanidium aminoglycodies, artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol (and thio analogs thereof),cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid,1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters(e.g., mono, bis, or tris fatty acid esters, e.g., C₁₀, C₁₁, C₁₂, C₁₃,C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ fatty acids) and ethers thereof,e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ alkyl;e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octaadecyl)glycerol),geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g.,gyceryl distearate), oleic acid, myristic acid,O3-(oleoyl)lithocholicacid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) andpeptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylatingagents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g.,imidazole, bisimidazole, histamine, imidazole clusters,acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles),dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the oligonucleotide agent into the cell, for example, bydisrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

The ligand can increase the uptake of the oligonucleotide agent into thecell by activating an inflammatory response, for example. Exemplaryligands that would have such an effect include tumor necrosis factoralpha (TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Alipid-based ligand can bind HSA with a sufficient affinity such that theconjugate will be preferably distributed to a non-kidney tissue.However, it is preferred that the affinity not be so strong that theHSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an I_(v)θ₃ integrin.Thus, one could use RGD peptides, cyclic peptides containing RGD, RGDpeptides that include D-amino acids, as well as synthetic RGD mimics. Inaddition to RGD, one can use other moieties that target the I_(v)-θ₃integrin ligand. Generally, such ligands can be used to controlproliferating cells and angiogeneis. Preferred conjugates of this typeinclude an oligonucleotide agent that targets PECAM-1, VEGF, or othercancer gene, e.g., a cancer gene described herein.

The oligonucleotide agents of the invention are particularly useful whentargeted to the liver. For example, a single stranded oligonucleotideagent featured in the invention can target an miRNA enriched in theliver, and the oligonucleotide agent can include a ligand for enhanceddelivery to the liver. An oligonucleotide agent can be targeted to theliver by incorporation of a monomer derivatized with a ligand whichtargets to the liver. For example, a liver-targeting agent can be alipophilic moiety. Preferred lipophilic moieties include lipid,cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilicmoieties that can function as liver-targeting agents include cholicacid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenicacid, dimethoxytrityl, or phenoxazine.

An oligonucleotide agent can also be targeted to the liver byassociation with a low-density lipoprotein (LDL), such as lactosylatedLDL. Polymeric carriers complexed with sugar residues can also functionto target oligonucleotide agents to the liver.

A targeting agent that incorporates a sugar, e.g., galactose and/oranalogues thereof, is particularly useful. These agents target, inparticular, the parenchymal cells of the liver. For example, a targetingmoiety can include more than one or preferably two or three galactosemoieties, spaced about 15 angstroms from each other. The targetingmoiety can alternatively be lactose (e.g., three lactose moieties),which is glucose coupled to a galactose. The targeting moiety can alsobe N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose ormannose-6-phosphate targeting moiety can be used for macrophagetargeting.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics tooligonucleotide agents can affect pharmacokinetic distribution of theiRNA, such as by enhancing cellular recognition and absorption. Thepeptide or peptidomimetic moiety can be about 5-50 amino acids long,e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long(see Table 3, for example).

TABLE 3 Exemplary Cell Permeation Peptides Cell Permeation PeptideAmino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKKDerossi et al., J. Biol. (SEQ ID NO: 31) Chem. 269:10444, 1994Tat fragment GRKKRRQRRRPPQC Vives et al., J. Biol. (48-60)(SEQ ID NO: 32) Chem., 272:16010, 1997 SignalGALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin et al., Sequence- (SEQ ID NO: 33)Biochem. Biophys. based peptide Res. Commun., 243:601, 1998 PVECLLIILRRRIRKQAHAHSK Elmquist et al., Exp. (SEQ ID NO: 34)Cell Res., 269:237, 2001 Transportan GWTLNSAGYLLKINLKALAALAKKILPooga et al., FASEB (SEQ ID NO:35) J., 12:67, 1998 AmphiphilicKLALKLALKALKAALKLA Oehlke et al., Mol. model peptide (SEQ ID NO: 36)Ther., 2:339, 2000 Arg₉ RRRRRRRRR Mitchell et al., J. (SEQ ID NO: 37)Pept. Res., 56:318, 2000 Bacterial cell KFFKFFKFFK wall (SEQ ID NO: 38)permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES (SEQ ID NO: 39)Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGP R (SEQ ID NO: 40) α-defensin ACYCRIPACIAGERRYGTCIYQGRLWAFC C (SEQ ID NO: 41) b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTCYR GKAKCCK (SEQ ID NO: 42) BactenecinRKCRIVVIRVCR (SEQ ID NO:43) PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (SEQ ID NO: 44) Indolicidin ILPWKWPWWPWRR-NH2(SEQ ID NO: 45)

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Thepeptide moiety can be an L-peptide or D-peptide. In another alternative,the peptide moiety can include a hydrophobic membrane translocationsequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGFhaving the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:46). An RFGFanalogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:47)containing a hydrophobic MTS can also be a targeting moiety. The peptidemoiety can be a “delivery” peptide, which can carry large polarmolecules including peptides, oligonucleotides, and protein across cellmembranes. For example, sequences from the HIV Tat protein(GRKKRRQRRRPPQ (SEQ ID NO:48) and the Drosophila Antennapedia protein(RQIKIWFQNRRMKWKK (SEQ ID NO:49) have been found to be capable offunctioning as delivery peptides. A peptide or peptidomimetic can beencoded by a random sequence of DNA, such as a peptide identified from aphage-display library, or one-bead-one-compound (OBOC) combinatoriallibrary (Lam et al., Nature 354:82-84, 1991). Preferably the peptide orpeptidomimetic tethered to an iRNA agent via an incorporated monomerunit is a cell targeting peptide such as an arginine-glycine-asparticacid (RGD)-peptide, or RGD mimic. A peptide moiety can range in lengthfrom about 5 amino acids to about 40 amino acids. The peptide moietiescan have a structural modification, such as to increase stability ordirect conformational properties. Any of the structural modificationsdescribed below can be utilized.

A “cell permeation peptide” is capable of permeating a cell, e.g.,a-mammalian cell, such as a human cell. A cell permeation peptide canalso include a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an SRMS can be anamphipathic a-helical peptide. Exemplary amphipathic a-helical peptidesinclude, but are not limited to, cecropins, lycotoxins, paradaxins,buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins,S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

Methods for Making iRNA Agents

iRNA agents conjugate to a lipophilic moiety for enhanced uptake intoneural cells can include modified or non-naturally occurring bases,e.g., bases described herein. In addition, iRNA agents can have amodified or non-naturally occurring base and another element describedherein.

The synthesis and purification of oligonucleotide peptide conjugates canbe performed by established methods. See, for example, Trufert et al.,Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugatesin Antisense Technology,” in Antisense Drug Technology, ed. S. T.Crooke, Marcel Dekker, Inc., 2001.

In one embodiment of the invention, a peptidomimetic can be modified tocreate a constrained peptide that adopts a distinct and specificpreferred conformation, which can increase the potency and selectivityof the peptide. For example, the constrained peptide can be anazapeptide (Gante, Synthesis 405-413, 1989). An azapeptide issynthesized by replacing the a-carbon of an amino acid with a nitrogenatom without changing the structure of the amino acid side chain. Forexample, the azapeptide can be synthesized by using hydrazine intraditional peptide synthesis coupling methods, such as by reactinghydrazine with a “carbonyl donor,” e.g., phenylchloroformate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an SRMS) can be an N-methylpeptide. N-methyl peptides are composed of N-methyl amino acids, whichprovide an additional methyl group in the peptide backbone, therebypotentially providing additional means of resistance to proteolyticcleavage. N-methyl peptides can by synthesized by methods known in theart (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99,2000; Cell Penetrating Peptides: Processes and Applications, Langel,ed., CRC Press, Boca Raton, Fla., 2002; Fische et al., Bioconjugate.Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002).For example, an Ant or Tat peptide can be an N-methyl peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an SRMS) can be a β-peptide.β-peptides form stable secondary structures such as helices, pleatedsheets, turns and hairpins in solutions. Their cyclic derivatives canfold into nanotubes in the solid state. β-peptides are resistant todegradation by proteolytic enzymes. β-peptides can be synthesized bymethods known in the art. For example, an Ant or Tat peptide can be aβ-peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an SRMS) can be an oligoureaconjugate (or an oligothiourea conjugate), in which the amide bond of apeptidomimetic is replaced with a urea moiety. Replacement of the amidebond provides increased resistance to degradation by proteolyticenzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In oneembodiment, an oligourea conjugate is tethered to an iRNA agent for usein oral delivery. The backbone in each repeating unit of an oligoureapeptidomimetic can be extended by one carbon atom in comparison with thenatural amino acid. The single carbon atom extension can increasepeptide stability and lipophilicity, for example. An oligourea peptidecan therefore be advantageous when an iRNA agent is directed for passagethrough a bacterial cell wall, or when an iRNA agent must traverse theblood-brain barrier, such as for the treatment of a neurologicaldisorder. In one embodiment, a hydrogen bonding unit is conjugated tothe oligourea peptide, such as to create an increased affinity with areceptor. For example, an Ant or Tat peptide can be an oligoureaconjugate (or an oligothiourea conjugate).

The dsRNA peptide conjugates of the invention can be affiliated with,e.g., tethered to, SRMSs occurring at various positions on an iRNAagent. For example, a peptide can be terminally conjugated, on eitherthe sense or the antisense strand, or a peptide can be bisconjugated(one peptide tethered to each end, one conjugated to the sense strand,and one conjugated to the antisense strand). In another option, thepeptide can be internally conjugated, such as in the loop of a shorthairpin iRNA agent. In yet another option, the peptide can be affiliatedwith a complex, such as a peptide-carrier complex.

A number of exemplary routes of delivery are described that can be usedto administer an oligonucleotide agent to a subject. In addition, theoligonucleotide agent can be formulated according to an exemplary methoddescribed herein.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an oligonucleotide agent (e.g., an oligonucleotide agent targeting anmiRNA or pre-miRNA) to tumors of a variety of other tissues, includingthe lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an oligonucleotide agent to the kidney. The RGD peptide can be linearor cyclic, and can be modified, e.g., glycosylated or methylated tofacilitate targeting to specific tissues. For example, a glycosylatedRGD peptide can deliver an oligonucleotide agent to a tumor cellexpressing α_(v)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an I_(v)θ₃ integrin.Thus, one could use RGD peptides, cyclic peptides containing RGD, RGDpeptides that include D-amino acids, as well as synthetic RGD mimics. Inaddition to RGD, one can use other moieties that target the I_(v)-θ₃integrin ligand. Generally, such ligands can be used to controlproliferating cells and angiogeneis. Preferred conjugates of this typeinclude an oligonucleotide agent that targets PECAM-1, VEGF, or othercancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to a ligand-conjugatedmonomer can be an amphipathic α-helical peptide. Exemplary amphipathicα-helical peptides include, but are not limited to, cecropins,lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP),cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinalantimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins,melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1,and caerins. A number of factors will preferably be considered tomaintain the integrity of helix stability. For example, a maximum numberof helix stabilization residues will be utilized (e.g., leu, ala, orlys), and a minimum number of helix destabilization residues will beutilized (e.g., proline, or cyclic monomeric units). The capping residuewill be considered (for example Gly is an exemplary N-capping residue)and/or C-terminal amidation can be used to provide an extra H-bond tostabilize the helix. Formation of salt bridges between residues withopposite charges, separated by i±3, or i±4 positions can providestability. For example, cationic residues such as lysine, arginine,homo-arginine, ornithine or histidine can form salt bridges with theanionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

In some embodiments, the peptide can have a cationic and/or ahydrophobic moiety.

In some embodiments, the ligand can be any of the nucleobases describedherein.

In some embodiments, the ligand can be a substituted amine, e.g.dimethylamino. In some embodiments, the substituted amine can bequaternized, e.g., by protonation or alkylation, rendering it cationic.In some embodiments, the substituted amine can be at the terminalposition of a relatively hydrophobic tether, e.g., alkylene.

In some embodiments, the ligand can be one of the following triterpenes:

In some embodiments, the ligand can be substituted or unsubstitutedcholesterol, or a stereoisomer thereof or one of the following steroids:

In some embodiments, a tethered ligand can contain one or more atomsthan the corresponding untethered or uncoupled ligand (e.g., one or moreprotons of a heteroatom-based functional group or an entireheteroatom-based functional group may be displaced from the uncoupledligand during coupling of a ligand to a carrier or tether). For example,the proton of the 3-hydroxy group of cholesterol can be replaced by atether (e.g., Chol-3-OH (uncoupled) and Chol-3-O-tether (coupled)) orthe entire 3-hydroxy group of cholesterol can be replaced by a sulfuratom (e.g., Chol-3-OH (uncoupled) and Chol-3-S-tether (coupled, e.g.,thiocholesterol)).

Methods for Making Oligonucleotide Agents

A listing of ribonucleosides containing the unusual bases describedherein are described in “The RNA Modification Database” maintained byPamela F. Crain, Jef Rozenski and James A. McCloskey; Departments ofMedicinal Chemistry and Biochemistry, University of Utah, Salt LakeCity, Utah 84112, USA.

The 5′ silyl protecting group can be used in conjunction with acidlabile orthoesters at the 2′ position of ribonucleosides to synthesizeoligonucleotides via phosphoramidite chemistry. Final deprotectionconditions are known not to significantly degrade RNA products.Functional groups on the unusual and universal bases are blocked duringoligonucleotide synthesis with protecting groups that are compatiblewith the operations being performed that are described herein. Allsyntheses can be conducted in any automated or manual synthesizer onlarge, medium, or small scale. The syntheses may also be carried out inmultiple well plates or glass slides.

The 5′-O-silyl group can be removed via exposure to fluoride ions, whichcan include any source of fluoride ion, e.g., those salts containingfluoride ion paired with inorganic counterions e.g., cesium fluoride andpotassium fluoride or those salts containing fluoride ion paired with anorganic counterion, e.g., a tetraalkylammonium fluoride. A crown ethercatalyst can be utilized in combination with the inorganic fluoride inthe deprotection reaction. Preferred fluoride ion source aretetrabutylammonium fluoride or aminehydrofluorides (e.g., combiningaqueous HF with triethylamine in a dipolar aprotic solvent, e.g.,dimethylformamide).

The choice of protecting groups for use on the phosphite triesters andphosphotriesters can alter the stability of the triesters towardsfluoride. Methyl protection of the phosphotriester or phosphitetriestercan stabilize the linkage against fluoride ions and improve processyields.

Since ribonucleosides have a reactive 2′ hydroxyl substituent, it can bedesirable to protect the reactive 2′ position in RNA with a protectinggroup that is compatible with a 5′-β-silyl protecting group, e.g. onestable to fluoride. Orthoesters meet this criterion and can be readilyremoved in a final acid deprotection step that can result in minimal RNAdegradation.

Tetrazole catalysts can be used in the standard phosphoramidite couplingreaction. Preferred catalysts include e.g. tetrazole, S-ethyl-tetrazole,p-nitrophenyltetrazole.

The general process is as follows. Nucleosides are suitably protectedand functionalized for use in solid-phase or solution-phase synthesis ofRNA oligonucleotides. The 2′-hydroxyl group in a ribonucleotide can bemodified using a tris orthoester reagent. The 2′-hydroxyl can bemodified to yield a 2′-O-orthoester nucleoside by reacting theribonucleoside with the tris orthoester reagent in the presence of anacidic catalyst, e.g., pyridinium p-toluene sulfonate. This reaction isknown to those skilled in the art. The product can then be subjected tofurther protecting group reactions (e.g., 5′-O-silylation) andfunctionalizations (e.g., 3′-O-phosphitylation) to produce a desiredreagent (e.g., nucleoside phosphoramidite) for incorporation within anoligonucleotide or polymer by reactions known to those skilled in theart.

Preferred orthoesters include those comprising ethylene glycol ligandswhich are protected with acyl or ester protecting groups. Specifically,the preferred acyl group is acetyl. The nucleoside reagents may then beused by those skilled in the art to synthesize RNA oligonucleotides oncommercially available synthesizer instruments, e.g., Gene AssemblerPlus (Pharmacia), 380B (Applied Biosystems). Following synthesis (eithersolution-phase or solid-phase) of an oligonucleotide or polymer, theproduct can be subjected to one or more reactions using non-acidicreagents. One of these reactions may be strong basic conditions, forexample, 40% methylamine in water for 10 minutes at 55° C., which willremove the acyl protecting groups from the ethylene glycol ligands butleave the orthoester moiety attached. The resultant orthoester may beleft attached when the polymer or oligonucleotide is used in subsequentapplications, or it may be removed in a final mildly-acidic reaction,for example, 10 minutes at 55° C. in 50 mM acetic acid, pH 3.0, followedby addition of equal volume of 150 mM TRIS buffer for 10 minutes at 55°C.

Universal bases are described in “Survey and Summary: The Applicationsof Universal DNA base analogues” Loakes, D., Nucleic Acid Research 2001,29, 2437, which is incorporated by reference in its entirety. Specificexamples are described in the following: Liu, D.; Moran, S.; Kool, E. T.Chem. Biol., 1997, 4, 919-926; Morales, J. C.; Kool, E. T. Biochemistry,2000, 39, 2626-2632; Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998,120, 6191-6192; Moran, S. Ren, R. X.-F.; Rumney IV, S.; Kool, E. T. J.Am. Chem. Soc., 1997, 119, 2056-2057; Guckian, K. M.; Morales, J. C.;Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656; Berger, M.; Wu. Y.;Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. NucleicAcids Res., 2000, 28, 2911-2914; Ogawa, A. K.; Wu, Y.; McMinn, D. L.;Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122,3274-3287; Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg,F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804; Tae, E. L.; Wu, Y.; Xia,G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123,7439-7440; Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P.G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632; McMinn, D.L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J.Am. Chem. Soc., 1999, 121, 11585-11586; Brotschi, C.; Haberli, A.;Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014; Weizman, H.;Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376; Lan, T.; McLaughlin, L.W. J. Am. Chem. Soc., 2000, 122, 6512-13.

As discussed above, the monomers and methods described herein can beused in the preparation of modified RNA molecules, or polymericmolecules comprising any combination of monomer compounds describedherein and/or natural or modified ribonucleotides in which one or moresubunits contain an unusual or universal base. Modified RNA moleculesinclude e.g. those molecules containing a chemically or stereochemicallymodified nucleoside (e.g., having one or more backbone modifications,e.g., phosphorothioate or P-alkyl; having one or more sugarmodifications, e.g., 2′-OCH₃ or 2′-F; and/or having one or more basemodifications, e.g., 5-alkylamino or 5-allylamino) or a nucleosidesurrogate.

Coupling of 5′-hydroxyl groups with phosphoramidites forms phosphiteester intermediates, which in turn are oxidized e.g., with iodine, tothe phosphate diester. Alternatively, the phosphites may be treatedwith, e.g., sulfur, selenium, amino, and boron reagents to form modifiedphosphate backbones. Linkages between the monomers described herein anda nucleoside or oligonucleotide chain can also be treated with iodine,sulfur, selenium, amino, and boron reagents to form unmodified andmodified phosphate backbones respectively. Similarly, the monomersdescribed herein may be coupled with nucleosides or oligonucleotidescontaining any of the modifications or nucleoside surrogates describedherein.

The synthesis and purification of oligonucleotide peptide conjugates canbe performed by established methods. See, for example, Trufert et al.,Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugatesin Antisense Technology,” in Antisense Drug Technology, ed. S. T.Crooke, Marcel Dekker, Inc., 2001.

In one embodiment of the invention, a peptidomimetic can be modified tocreate a constrained peptide that adopts a distinct and specificpreferred conformation, which can increase the potency and selectivityof the peptide. For example, the constrained peptide can be anazapeptide (Gante, Synthesis, 1989, 405-413). An azapeptide issynthesized by replacing the α-carbon of an amino acid with a nitrogenatom without changing the structure of the amino acid side chain. Forexample, the azapeptide can be synthesized by using hydrazine intraditional peptide synthesis coupling methods, such as by reactinghydrazine with a “carbonyl donor,” e.g., phenylchloroformate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an ligand-conjugated monomer) canbe an N-methyl peptide. N-methyl peptides are composed of N-methyl aminoacids, which provide an additional methyl group in the peptide backbone,thereby potentially providing additional means of resistance toproteolytic cleavage. N-methyl peptides can by synthesized by methodsknown in the art (see, for example, Lindgren et al., Trends Pharmacol.Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications,Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al.,Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc.,124:13382, 2002). For example, an Ant or Tat peptide can be an N-methylpeptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe a β-peptide. β-peptides form stable secondary structures such ashelices, pleated sheets, turns and hairpins in solutions. Their cyclicderivatives can fold into nanotubes in the solid state. β-peptides areresistant to degradation by proteolytic enzymes. β-peptides can besynthesized by methods known in the art. For example, an Ant or Tatpeptide can be a β-peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe a oligocarbamate. Oligocarbamate peptides are internalized into acell by a transport pathway facilitated by carbamate transporters. Forexample, an Ant or Tat peptide can be an oligocarbamate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe an oligourea conjugate (or an oligothiourea conjugate), in which theamide bond of a peptidomimetic is replaced with a urea moiety.Replacement of the amide bond provides increased resistance todegradation by proteolytic enzymes, e.g., proteolytic enzymes in thegastrointestinal tract. In one embodiment, an oligourea conjugate istethered to an oligonucleotide agent for use in oral delivery. Thebackbone in each repeating unit of an oligourea peptidomimetic can beextended by one carbon atom in comparison with the natural amino acid.The single carbon atom extension can increase peptide stability andlipophilicity, for example. An oligourea peptide can therefore beadvantageous when an oligonucleotide agent is directed for passagethrough a bacterial cell wall, or when an oligonucleotide agent musttraverse the blood-brain barrier, such as for the treatment of aneurological disorder. In one embodiment, a hydrogen bonding unit isconjugated to the oligourea peptide, such as to create an increasedaffinity with a receptor. For example, an Ant or Tat peptide can be anoligourea conjugate (or an oligothiourea conjugate).

The siRNA peptide conjugates of the invention can be affiliated with,e.g., tethered to, ligand-conjugated monomers occurring at variouspositions on an oligonucleotide agent. For example, a peptide can beterminally conjugated, on either the sense or the antisense strand, or apeptide can be bisconjugated (one peptide tethered to each end, oneconjugated to the sense strand, and one conjugated to the antisensestrand). In another option, the peptide can be internally conjugated,such as in the loop of a short hairpin oligonucleotide agent. In yetanother option, the peptide can be affiliated with a complex, such as apeptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, whichcan encapsulate one or more oligonucleotide agents (such as for deliveryto a biological system and/or a cell), and a peptide moiety tethered tothe outside of the carrier molecule, such as for targeting the carriercomplex to a particular tissue or cell type. A carrier complex can carryadditional targeting molecules on the exterior of the complex, orfusogenic agents to aid in cell delivery. The one or moreoligonucleotide agents encapsulated within the carrier can be conjugatedto lipophilic molecules, which can aid in the delivery of the agents tothe interior of the carrier.

A carrier molecule or structure can be, for example, a micelle, aliposome (e.g., a cationic liposome), a nanoparticle, a microsphere, ora biodegradable polymer. A peptide moiety can be tethered to the carriermolecule by a variety of linkages, such as a disulfide linkage, an acidlabile linkage, a peptide-based linkage, an oxyamino linkage or ahydrazine linkage. For example, a peptide-based linkage can be a GFLGpeptide. Certain linkages will have particular advantages, and theadvantages (or disadvantages) can be considered depending on the tissuetarget or intended use. For example, peptide based linkages are stablein the blood stream but are susceptible to enzymatic cleavage in thelysosomes.

The protected monomer compounds can be separated from a reaction mixtureand further purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Other synthetic chemistry transformations, protecting groups(e.g., for hydroxyl, amino, etc. present on the bases) and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The protected monomer compounds of this invention may contain one ormore asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of these compounds areexpressly included in the present invention. The compounds describedherein can also contain linkages (e.g., carbon-carbon bonds,carbon-nitrogen bonds, e.g., amides) or substituents that can restrictbond rotation, e.g. restriction resulting from the presence of a ring ordouble bond. Accordingly, all cis/trans, E/Z isomers, and rotationalisomers (rotamers) are expressly included herein. The compounds of thisinvention may also be represented in multiple tautomeric forms, in suchinstances, the invention expressly includes all tautomeric forms of thecompounds described herein (e.g., alkylation of a ring system may resultin alkylation at multiple sites, the invention expressly includes allsuch reaction products). All such isomeric forms of such compounds areexpressly included in the present invention. All crystal forms of thecompounds described herein are expressly included in the presentinvention.

Representative ligand-conjugated monomers and typical syntheses forpreparing ligand-conjugated monomers and related compounds describedherein are provided below. As discussed elsewhere, protecting groups forligand-conjugated monomer hydroxyl groups, e.g., OFG¹, include but arenot limited to the dimethoxytrityl group (DMT). For example, it can bedesirable in some embodiments to use silicon-based protecting groups asa protecting group for OFG¹. Silicon-based protecting groups cantherefore be used in conjunction with or in place of the DMT group asnecessary or desired. Thus, the ligand-conjugated monomers and synthesesdelineated below, which feature the DMT protecting group as a protectinggroup for OFG¹, is not to be construed as limiting in any way to theinvention.

Synthesis of Pyrroline Carrier

Synthesis of 5′-labelled siRNA

25 & 26 can be used for 3′,5′-conjugation respectively.

Synthesis of Pthalimido Derivative

30 and 31 can be converted to similar derivatives as shown in schemes2-4 for 3′ and 5′ cpnjugation of siRNA

Synthesis of Thalimido Derivative

40 and 41 can be converted to similar derivatives as shown in schemes2-4 for 3′ and 5′ cpnjugation of siRNA

Synthesis of N-alkyl pyrroline derivatives

Intermediates 50 and 51 can be converted to analogs which could beconjugated with siRNA using similar reactions

Piperidine Series Ligands:

Similar to pyrroline series piperidine series can be synthesised

Piperidine Series Ligands:

Similar to pyrroline series piperidine series can be synthesised

Hydroxy Proline Series Linkers:

From commercially available cis-3-hydroxy proline and (s)-pyrrolidonecarboxylate

Phthalimide Derivative to Stabilise siRNA

4-hydroxy Proline Derivatives

Phthalimido Derivatives

Synthesis of 6-membered Linker

Simliar reaction can be carried out with 2-piperidone and 3-piperidone

Linkers from 4-piperidone

Linkers from 3-piperidone

Linkers from 2-piperidone

Conjugation through Decalin System

Conjugates from Decalin System:

Decalin Linker from Wieland-Miescher Ketone

Conjugates from Wieland-Miescher Ketone

Synthesis of Pyrroline Linker:

Solid Phase Synthesis and Post-Synthesis Conjugation:

Exemplary Ligand Conjugated Monomers LCM-E.g.-

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6.

7.

8.

Conjugation of Ligands to Oligonucleotide Agents

The conjugation of a ligand to an oligonucleotide agent, e.g., anoligonucleotide agent that targets an miRNA or pre-miRNA can have afavorable effect on the modulating effect of the agent. For example, theagent can improve pharmacokinetics, stability, and/or tissuespecificity.

In some embodiments, an oligonucleotide agent (referred to as “NA” informula OT-I through OT-IV below, e.g., RNA, DNA, chimeric RNA-DNA,DNA-RNA, RNA-DNA-RNA, or DNA-RNA-DNA) can be chemically modified byconjugating a moiety that includes a ligand having one or more chemicallinkages for attachment of the ligand (L) to the oligonucleotide ornucleic acid. The ligand of an oligonucleotide agent can be coupled byone or both of a tether and linker. In the diagram below, exemplarychemical linkages are represented as X, Y, and Z. These can be part ofthe tether or linker.

Ligands can be attached at one or both of the 3′ end, the 5′ end, andinternal positions. In certain embodiments, the oligonucleotide agentcan be chemically modified by conjugating one or more moieties havingformula OT-I. Table 4, shows a variety of conjugates.

TABLE 4

Exemplary ligands are listed in Table 5 and are discussed elsewhereherein. The exemplary ligands (L) shown in Table 5 are preferred.

TABLE 5

L = Cholesterol Thiocholesterol 5β-Cholanic Acid Cholic acid Lithocholicacid Biotin Vitamin E Naproxen Ibuprofen Amines (mono, di, tri,tetraalkyl or aryl) Folate Sugar (N-Acetylgalactosamine, galactosamine,galactose, Mannose) —(CH₂)_(n)NQ₁Q₂, where n = 0-40, Q₁, Q₂ = H, Me orEt; Q₁ = H, Q₂ = H, Me, Et or aryl —(CH₂)_(p)CH═CH(CH₂)_(q)NQ₁Q₂, wherep and/or q = 0-40, Q₁, Q₂ = H, Me or Et; Q₁= H, Q₂ = H, Me, Et or arylwith E and/or Z configuration —(CH₂)_(p)CH═CH(CH₂)_(q)NQ₁Q₂, where pand/or q = 0-40, Q₁, Q₂ = H, Me or Et; Q₁ = H, Q₂ = H, Me, Et or aryl—(CH₂)_(p)CH═CH(CH₂)_(q)CH═CH(CH₂)_(r)NQ₁Q₂, where p, q and/or r = 0-40,Q₁, Q₂ = H, Me or Et; Q₁ = H, Q₂ = H, Me, Et or aryl with E and/or Zconfiguration —O(CH₂)_(m)(OCH₂CH₂)_(n)—OR, where m, n = 0-40 and R = H,Me, NQ₁Q₂, —C(O)NR′R″ —C(S)NR′R″ —NH(CH₂)_(m)(OCH₂CH₂)_(n)—OR, where m,n = 0-40 and R = H, Me, NQ₁Q₂, —C(O)NR′R″ —C(S)NR′R″—(CH₂)_(m)(NHCH₂CH₂)_(n)—R, where m, n = 0-40 and R = H, OH, Me, NQ₁Q₂,—C(O)NR′R″ —C(S)NR′R″ —NH(CH₂)_(m)(NHCH₂CH₂)_(n)—R, where m, n = 0-40and R = H, OH, Me, NQ₁Q₂, —C(O)NR′R″ —C(S)NR′R″ Dialkylglycerol (sn3,sn1, sn2 and racemic) with number of methylene varies from 0-40Dlacylglycerol (sn3, sn1, sn2 and racemic) with number of methylenevaries from 0-40 Dialkylglycerol (sn3, sn1, sn2 and racemic) with numberof methylene varies from 0-40 and the alkyl chian contains one or moredouble bonds with E and/or Z isomers Dlacylglycerol (sn3, snl , sn2 andracemic) with number of methylene varies from 0-40 and the alkyl chiancontains one or more double bonds with E and/or Z isomers Lipids

Exemplary X, Y, and Z moieties are shown in in Table 6. The X, Y, and Zmoieties can be selected independently of one another.

TABLE 6

X = —NHC(O)— Y = —NHC(O)— Z = —NHC(O)— —C(O)NH— —C(O)NH— —C(O)NH——OC(O)NH— —OC(O)NH— —OC(O)NH— —NHC(O)O— —NHC(O)O— —NHC(O)O— —O— —O— —O——S— —S— —S— —SS— —SS— —SS— —S(O)— —S(O)— —S(O)— —S(O₂)— —S(O₂)— —S(O₂)——NHC(O)NH— —NHC(O)NH— —NHC(O)NH— —NHC(S)NH— —NHC(S)NH— —NHC(S)NH——C(O)O— —C(O)O— —C(O)O— —OC(O)— —OC(O)— —OC(O)— —NHC(S)— —NHC(S)——NHC(S)— —NHC(S)O— —NHC(S)O— —NHC(S)O— —C(S)NH— —C(S)NH— —C(S)NH——OC(S)NH— —OC(S)NH— —OC(S)NH— —NHC(S)O— —NHC(S)O— —NHC(S)O— —CH₂— —CH₂——CH₂— —CH₂CH═CH— —CH₂CH═CH— —CH₂CH═CH— —C(O)CH═CH— —C(O)CH═CH——C(O)CH═CH— —NH—CH₂CH═CH— —NH—CH₂CH═CH— —NH—CH₂CH═CH— —O—P(O)(OH)—O——O—P(O)(OH)—O— —O—P(O)(OH)—O— —O—P(S)(OH)—O— —O—P(S)(OH)—O——O—P(S)(OH)—O— —O—P(S)(SH)—O— —O—P(S)(SH)—O— —O—P(S)(SH)—O——S—P(O)(OH)—O— —S—P(O)(OH)—O— —S—P(O)(OH)—O— —O—P(O)(OH)—S——O—P(O)(OH)—S— —O—P(O)(OH)—S— —S—P(O)(OH)—S— —S—P(O)(OH)—S——S—P(O)(OH)—S— —O—P(S)(OH)—S— —O—P(S)(OH)—S— —O—P(S)(OH)—S——S—P(S)(OH)—O— —S—P(S)(OH)—O— —S—P(S)(OH)—O— —O—P(O)(R)—O— —O—P(O)(R)—O——O—P(O)(R)—O— —O—P(S)(R)—O— —O—P(S)(R)—O— —O—P(S)(R)—O— —S—P(O)(R)—O——S—P(O)(R)—O— —S—P(O)(R)—O— —S—P(S)(R)—O— —S—P(S)(R)—O— —S—P(S)(R)—O——S—P(O)(R)—S— —S—P(O)(R)—S— —S—P(O)(R)—S— —O—P(S)(R)—S— —O—P(S)(R)—S——O—P(S)(R)—S— R = Alkyl, fluroalkyl, aryl or aralkyl

Exemplary tethers are shown in Table 7.

Linker = 3′-end 5′-end interior Tether: —(CH₂)_(n)—, where n = 1-40—(CH₂—CH₂O)_(n)—, where n = 1-20 —O(CH₂—CH₂O)_(n)—, where n = 1-20—(CH₂—CH₂NH)_(n)—, where n = 1-20 —NH(CH₂—CH₂NH)_(n)—, where n = 1-20—(CH₂)_(l)[(CH═CH)_(m)(CH₂)_(n)]_(p)(CH═CH)_(q)(CH₂)_(r)—, where l, m,n, p, q and/or r = 0-20—(CH₂)_(l)[(C≡C)_(m)(CH₂)_(n)]_(p)(C≡C)_(q)(CH₂)_(r)—, where l, m, n, p,q and/or r = 0-20

Compounds described herein can be prepared by methods described hereinor by conventional methods from commercially available reagents andstarting materials.

Compound I is prepared as reported by Fraser et al. (Tetrahedron Lett.41:1523, 2000). Steps (ii), (iii) (a), (iii) (c), (iv), (v) and (vii)are performed according to literature procedure (Fraser et al.,Tetrahedron Lett. 41:1523, 2000). Step (iii) (b) and (v) (b) areperformed as reported in the literature (Bioorg. Med. Chem. Lett.13:1713, 2003). Step (iv) is performed as reported in the literature(Corey and Venkateswarlu, J. Am. Chem. Soc. 94:6190, 1972).

The synthesis of certain compounds is described in scheme 2, below. Step(i) is performed as reported in Dubowchik and Radia (Tetrahedron Lett.,38:5257, 1997); step (ii) is performed as reported in Corey andVenkateswarlu (J. Am. Chem. Soc. 94:6190, 1972); step (iii) is performedas reported in Fraser et al. (Tetrahedron Lett. 41:1523, 2000) and step(iv) is performed as described in Miller et al. (Current Protocol inNucleic Acids Chemistry, 2000, 2.5.1-2.5.36, John Wiley and Sons, Inc.).

The synthesis of certain compounds is performed as described in Scheme3, below. Step (i) is performed as described in Miller et al. (CurrentProtocol in Nucleic Acids Chemistry, 2000, 2.5.1-2.5.36, John Wiley andSons, Inc.); step (ii) is performed as reported in the Corey andVenkateswarlu (J. Am. Chem. Soc. 94:6190, 1972) and step (iii) isperformed as reported by Fraser et al. (Tetrahedron Lett. 41:1523,2000).

The synthesis of certain compounds is performed as described in Scheme 4below. Step (ii) is performed as reported in Corey and Venkateswarlu (J.Am. Chem. Soc. 94:6190, 1972) and step (iii) is performed as reported byFraser et al. (Tetrahedron Lett. 41:1523, 2000).

The synthesis of certain compounds is described in Scheme 5, below. Step(i) is performed as described in Miller et al. (Current Protocol inNucleic Acids Chemistry, 2000, 2.5.1-2.5.36, John Wiley and Sons, Inc.);step (ii) is performed as described in Corey and Venkateswarlu (J. Am.Chem. Soc. 94:6190, 1972) and step (iii) is performed as reported byFraser et al. (Tetrahedron Lett. 41:1523, 2000).

The synthesis of certain compounds is described in Scheme 6, below.Compound 130, shown in Scheme 6, is obtained as reported in Liu andAustin, J. Org. Chem. 66:8643, 2001). Step (i) and (iii) (b) areperformed as reported in the literature (Chem. Rev., 1954, 54, 1); step(ii) (a) is performed according to literature procedures (J. Org. Chem.,1993, 58, 2334); step (ii) (b), (iii) (a) and (iv) (b) are performed asreported in the literature (Bioorg. Med. Chem. Lett., 2003, 13, 1713);step (iii) (c) is performed as reported in Dubowchik and Radia(Tetrahedron Lett. 38:5257, 1997); step (iv) (a) is performed asreported in the literature (Organic Lett., 2001, 3, 1809); step (v) isperformed as reported in Corey and Venkateswarlu (J. Am. Chem. Soc.94:6190, 1972) and step (vi) is performed as reported by Fraser et al.(Tetrahedron Lett. 41:1523, 2000).

The synthesis of certain compounds is described in Scheme 7, below.Compound 146 is obtained as reported in Liu and Austin (J. Org. Chem.,2001, 66, 8643). Step (i) (b) and (iii) (c) are performed as reported inthe literature (Chem. Rev., 1954, 54, 1); step (ii) (a) is performedaccording to literature procedures (J. Org. Chem., 1993, 58, 2334); step(ii) (b), (iii) (b) and (iv) (b) are performed as reported in theliterature (Bioorg. Med. Chem. Lett., 2003, 13, 1713); step (iii) (d) isperformed as reported in Dubowchik and Radia (Tetrahedron Lett., 1997,38, 5257); step (iv) (a) is performed as reported in the literature(Organic Lett., 2001, 3, 1809); step (v) is performed as reported inCorey and Venkateswarlu (J. Am. Chem. Soc., 1972, 94, 6190) and step(vi) is performed as reported by Fraser et al. (Tetrahedron Lett., 2000,41, 1523)

The synthesis of certain compounds is described in Scheme 8, below.Compound 163 is obtained as reported in Liu and Austin (J. Org. Chem.,2001, 66, 8643).

The synthesis of certain compounds is described in Scheme 9, below.Compound 180 is obtained as reported in Liu and Austin (J. Org. Chem.,2001, 66, 8643).

Ligand-conjugated Monomer Subunits and Monomers for OligonucleotideSynthesis Definitions

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may beoptionally inserted with O, N, or S. The terms “aralkyl” refers to analkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “aralkyl” include benzyl,9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-8 carbon atoms and characterized in having one or moredouble bonds. Examples of a typical alkenyl include, but not limited to,allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term“alkynyl” refers to a straight or branched hydrocarbon chain containing2-8 carbon atoms and characterized in having one or more triple bonds.Some examples of a typical alkynyl are ethynyl, 2-propynyl, and3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionallyserve as the point of attachment of the alkenyl and alkynyl groups,respectively.

The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and—NH(alkyl)₂ radicals respectively. The term “aralkylamino” refers to a—NH(aralkyl) radical. The term “alkoxy” refers to an —O-alkyl radical,and the terms “cycloalkoxy” and “aralkoxy” refer to an —O-cycloalkyl andO-aralkyl radicals respectively. The term “siloxy” refers to a R₃SiO—radical.

The term “mercapto” refers to an SH radical. The term “thioalkoxy”refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalentspecies of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom can be substituted. The cycloalkyl groupsherein described may also contain fused rings. Fused rings are ringsthat share a common carbon-carbon bond or a common carbon atom (e.g.,spiro-fused rings). Examples of cycloalkyl moieties include, but are notlimited to, cyclohexyl, adamantyl, and norbornyl, and decalin.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom can be substituted. The heterocyclyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of heterocyclyl include, but are not limited totetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl and pyrrolidinyl.

The term “cycloalkenyl” as employed herein includes partiallyunsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or polycyclichydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons,wherein any ring atom can be substituted. The cycloalkenyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of cycloalkenyl moieties include, but are not limitedto cyclohexenyl, cyclohexadienyl, or norbornenyl.

The term “heterocycloalkenyl” refers to a partially saturated,nonaromatic 5-10 membered monocyclic, 8-12 membered bicyclic, or 11-14membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, saidheteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6,or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,respectively), wherein any ring atom can be substituted. Theheterocycloalkenyl groups herein described may also contain fused rings.Fused rings are rings that share a common carbon-carbon bond or a commoncarbon atom (e.g., spiro-fused rings). Examples of heterocycloalkenylinclude but are not limited to tetrahydropyridyl and dihydropyran.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2),amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, andcombinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” andthe like refer to radicals of adenine, cytosine, guanine, thymine, anduracil.

A “protected” moiety refers to a reactive functional group, e.g., ahydroxyl group or an amino group, or a class of molecules, e.g., sugars,having one or more functional groups, in which the reactivity of thefunctional group is temporarily blocked by the presence of an attachedprotecting group. Protecting groups useful for the monomers and methodsdescribed herein can be found, e.g., in Greene, T. W., Protective Groupsin Organic Synthesis (John Wiley and Sons: New York), 1981, which ishereby incorporated by reference.

As used herein, an “unusual” nucleobase can include any one of thefollowing:

-   2-methyladeninyl,-   N6-methyladeninyl,-   2-methylthio-N-6-methyladeninyl,-   N6-isopentenyladeninyl,-   2-methylthio-N-6-isopentenyladeninyl,-   N6-(cis-hydroxyisopentenyl)adeninyl,-   2-methylthio-N-6-(cis-hydroxyisopentenyl) adeninyl,-   N6-glycinylcarbamoyladeninyl,-   N6-threonylcarbamoyladeninyl,-   2-methylthio-N-6-threonyl carbamoyladeninyl,-   N6-methyl-N-6-threonylcarbamoyladeninyl,-   N6-hydroxynorvalylcarbamoyladeninyl,-   2-methylthio-N-6-hydroxynorvalyl carbamoyladeninyl,-   N6,N6-dimethyladeninyl,-   3-methylcytosinyl,-   5-methylcytosinyl,-   2-thiocytosinyl,-   5-formylcytosinyl,

-   N4-methylcytosinyl,-   5-hydroxymethylcytosinyl,-   1-methylguaninyl,-   N2-methylguaninyl,-   7-methylguaninyl,-   N2,N2-dimethylguaninyl,

-   N2,7-dimethylguaninyl,-   N2,N2,7-trimethylguaninyl,-   1-methylguaninyl,-   7-cyano-7-deazaguaninyl,-   7-aminomethyl-7-deazaguaninyl,-   pseudouracilyl,-   dihydrouracilyl,-   5-methyluracilyl,-   1-methylpseudouracilyl,-   2-thiouracilyl,-   4-thiouracilyl,-   2-thiothyminyl-   5-methyl-2-thiouracilyl,-   3-(3-amino-3-carboxypropyl)uracilyl,-   5-hydroxyuracilyl,-   5-methoxyuracilyl,-   uracilyl 5-oxyacetic acid,-   uracilyl 5-oxyacetic acid methyl ester,-   5-(carboxyhydroxymethyl)uracilyl,-   5-(carboxyhydroxymethyl)uracilyl methyl ester,-   5-methoxycarbonylmethyluracilyl,-   5-methoxycarbonylmethyl-2-thiouracilyl,-   5-aminomethyl-2-thiouracilyl,-   5-methylaminomethyluracilyl,-   5-methylaminomethyl-2-thiouracilyl,-   5-methylaminomethyl-2-selenouracilyl,-   5-carbamoylmethyluracilyl,-   5-carboxymethylaminomethyluracilyl,-   5-carboxymethylaminomethyl-2-thiouracilyl,-   3-methyluracilyl,-   1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,-   5-carboxymethyluracilyl,-   5-methyldihydrouracilyl, or-   3-methylpseudouracilyl.

A universal base can form base pairs with each of the natural DNA/RNAbases, exhibiting relatively little discrimination between them. Ingeneral, the universal bases are non-hydrogen bonding, hydrophobic,aromatic moieties which can stabilize e.g., duplex RNA or RNA-likemolecules, via stacking interactions. A universal base can also includehydrogen bonding substituents.

General

An oligonucleotide agent, e.g., a conjugated oligonucleotide agent,containing a preferred, but nonlimiting ligand-conjugated monomersubunit is presented as formula (II) below. The carrier (also referredto in some embodiments as a “linker”) can be a cyclic or acyclic moietyand includes two “backbone attachment points” (e.g., hydroxyl groups)and a ligand. The ligand can be directly attached (e.g., conjugated) tothe carrier or indirectly attached (e.g., conjugated) to the carrier byan intervening tether (e.g., an acyclic chain of one or more atoms; or anucleobase, e.g., a naturally occurring nucleobase optionally having oneor more chemical modifications, e.g., an unusual base; or a universalbase). The carrier therefore also includes a “ligand or tetheringattachment point” for the ligand and tether/tethered ligand,respectively.

The ligand-conjugated monomer subunit may be the 5′ or 3′ terminalsubunit of the RNA molecule, i.e., one of the two “W” groups may be ahydroxyl group, and the other “W” group may be a chain of two or moreunmodified or modified ribonucleotides. Alternatively, theligand-conjugated monomer subunit may occupy an internal position, andboth “W” groups may be one or more unmodified or modifiedribonucleotides. More than one ligand-conjugated monomer subunit may bepresent in a RNA molecule, e.g., an oligonucleotide agent. Preferredpositions for inclusion of a tethered ligand-conjugated monomer subunit,e.g., one in which a lipophilic moiety, e.g., cholesterol, is tetheredto the carrier are at the 3′ terminus, the 5′ terminus, or at aninternal position.

The modified RNA molecule of formula (II) can be obtained usingoligonucleotide synthetic methods known in the art. In a preferredembodiment, the modified RNA molecule of formula (II) can be prepared byincorporating one or more of the corresponding monomer compounds (see,e.g., A, B, and C below) into a growing strand, utilizing, e.g.,phosphoramidite or H-phosphonate coupling strategies.

The monomers, e.g., a ligand-conjugated monomers, generally include twodifferently functionalized hydroxyl groups (OFG¹ and OFG²), which arelinked to the carrier molecule (see A below), and a ligand/tetheringattachment point. As used herein, the term “functionalized hydroxylgroup” means that the hydroxyl proton has been replaced by anothersubstituent. As shown in representative structures B and C below, onehydroxyl group (OFG¹) on the carrier is functionalized with a protectinggroup (PG). The other hydroxyl group (OFG²) can be functionalized witheither (1) a liquid or solid phase synthesis support reagent (solidcircle) directly or indirectly through a linker, L, as in B, or (2) aphosphorus-containing moiety, e.g., a phosphoramidite as in C. Thetethering attachment point may be connected to a hydrogen atom, asuitable protecting group, a tether, or a tethered ligand at the timethat the monomer is incorporated into the growing strand (see variable“R” in A below). Thus, the tethered ligand can be, but need not beattached to the monomer at the time that the monomer is incorporatedinto the growing strand. In certain embodiments, the tether, the ligandor the tethered ligand may be linked to a “precursor” ligand-conjugatedmonomer subunit after a “precursor” ligand-conjugated monomer subunithas been incorporated into the strand. The wavy line used below (andelsewhere herein) refers to a connection, and can represent a directbond between the moiety and the attachment point or a tethering moleculewhich is interposed between the moiety and the attachment point.Directly tethered means the moiety is bound directly to the attachmentpoint. Indirectly tethered means that there is a tether moleculeinterposed between the attachment point and the moiety.

The (OFG¹) protecting group may be selected as desired, e.g., from T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991). The protecting group is preferablystable under amidite synthesis conditions, storage conditions, andoligonucleotide synthesis conditions. Hydroxyl groups, —OH, arenucleophilic groups (i.e., Lewis bases), which react through the oxygenwith electrophiles (i.e., Lewis acids). Hydroxyl groups in which thehydrogen has been replaced with a protecting group, e.g., atriarylmethyl group or a trialkylsilyl group, are essentially unreactiveas nucleophiles in displacement reactions. Thus, the protected hydroxylgroup is useful in preventing e.g., homocoupling of compoundsexemplified by structure C during oligonucleotide synthesis. In someembodiments, a preferred protecting group is the dimethoxytrityl group.In other embodiments, a preferred protecting group is a silicon-basedprotecting group having the formula below:

X⁵′, X⁵″, and X⁵′″ can be selected from substituted or unsubstitutedalkyl, cycloalkyl, aryl, araklyl, heteroaryl, alkoxy, cycloalkoxy,aralkoxy, aryloxy, heteroaryloxy, or siloxy (i.e., R₃SiO—, the three “R”groups can be any combination of the above listed groups). X^(5′),X^(5″), and X^(5′″) may all be the same or different; also contemplatedis a combination in which two of X^(5′), X^(5″), and X^(5′″) areidentical and the third is different. In certain embodiments X^(5′),X^(5″), and X^(5′″) include at least one alkoxy or siloxy groups. Apreferred combination includes X^(5′), X^(5″)=trimethylsiloxy andX^(5′″)=1,3-(tri-2-propoxy or cyclododecyloxy.

Other preferred combinations of X^(5′), X^(5″), and X^(5′″) includethose that result in OFG¹ groups that meet the deprotection andstability criteria delineated below. The group is preferably stableunder amidite synthesis conditions, storage conditions, andoligonucleotide synthesis conditions. Rapid removal, i.e., less than oneminute, of the silyl group from e.g., a support-bound oligonucleotide isdesirable because it can reduce synthesis times and thereby reduceexposure time of the growing oligonucleotide chain to the reagents.Oligonucleotide synthesis can be improved if the silyl protecting groupis visible during deprotection, e.g., from the addition of a chromophoresilyl substituent.

Selection of silyl protecting groups can be complicated by the competingdemands of the essential characteristics of stability and facileremoval, and the need to balance these competitive goals. Mostsubstituents that increase stability can also increase the reaction timerequired for removal of the silyl group, potentially increasing thelevel of difficulty in removal of the group.

The addition of alkoxy and siloxy substituents to OFG¹silicon-containing protecting groups increases the susceptibility of theprotecting groups to fluoride cleavage of the silylether bonds.Increasing the steric bulk of the substituents preserves stability whilenot decreasing fluoride lability to an equal extent. An appropriatebalance of substituents on the silyl group makes a silyl ether a viablenucleoside protecting group.

Candidate OFG¹ silicon-containing protecting groups may be tested byexposing a tetrahydrofuran solution of a preferred carrier bearing thecandidate OFG¹ group to five molar equivalents of tetrahydrofuran atroom temperature. The reaction time may be determined by monitoring thedisappearance of the starting material by thin layer chromatography.

When the OFG² in B includes a linker, e.g., a relatively long organiclinker, connected to a soluble or insoluble support reagent, solution orsolid phase synthesis techniques can be employed to build up a chain ofnatural and/or modified ribonucleotides once OFG¹ is deprotected andfree to react as a nucleophile with another nucleoside or monomercontaining an electrophilic group (e.g., an amidite group).Alternatively, a natural or modified ribonucleotide oroligoribonucleotide chain can be coupled to monomer C via an amiditegroup or H-phosphonate group at OFG². Subsequent to this operation, OFG¹can be deblocked, and the restored nucleophilic hydroxyl group can reactwith another nucleoside or monomer containing an electrophilic group. R′can be substituted or unsubstituted alkyl or alkenyl. In preferredembodiments, R′ is methyl, allyl or 2-cyanoethyl. R″ may a C₁-C₁₀ alkylgroup, preferably it is a branched group containing three or morecarbons, e.g., isopropyl.

OFG² in B can be hydroxyl functionalized with a linker, which in turncontains a liquid or solid phase synthesis support reagent at the otherlinker terminus. The support reagent can be any support medium that cansupport the monomers described herein. The monomer can be attached to aninsoluble support via a linker, L, which allows the monomer (and thegrowing chain) to be solubilized in the solvent in which the support isplaced. The solubilized, yet immobilized, monomer can react withreagents in the surrounding solvent; unreacted reagents and solubleby-products can be readily washed away from the solid support to whichthe monomer or monomer-derived products is attached. Alternatively, themonomer can be attached to a soluble support moiety, e.g., polyethyleneglycol (PEG) and liquid phase synthesis techniques can be used to buildup the chain. Linker and support medium selection is within skill of theart. Generally the linker may be —C(O)(CH₂)_(q)C(O)—, or—C(O)(CH₂)_(q)S—, in which q can be 0, 1, 2, 3, or 4; preferably, it isoxalyl, succinyl or thioglycolyl. Standard control pore glass solidphase synthesis supports can not be used in conjunction with fluoridelabile 5′ silyl protecting groups because the glass is degraded byfluoride with a significant reduction in the amount of full-lengthproduct. Fluoride-stable polystyrene based supports or PEG arepreferred.

The ligand/tethering attachment point can be any divalent, trivalent,tetravalent, pentavalent or hexavalent atom. In some embodiments,ligand/tethering attachment point can be a carbon, oxygen, nitrogen orsulfur atom. For example, a ligand/tethering attachment point precursorfunctional group can have a nucleophilic heteroatom, e.g., —SH, —NH₂,secondary amino, ONH₂, or NH₂NH₂. As another example, theligand/tethering attachment point precursor functional group can be anolefin, e.g., —CH═CH₂ or a Diels-Alder diene or dienophile and theprecursor functional group can be attached to a ligand, a tether, ortethered ligand using, e.g., transition metal catalyzed carbon-carbon(for example olefin metathesis) processes or cycloadditions (e.g.,Diels-Alder). As a further example, the ligand/tethering attachmentpoint precursor functional group can be an electrophilic moiety, e.g.,an aldehyde. When the carrier is a cyclic carrier, the ligand/tetheringattachment point can be an endocyclic atom (i.e., a constituent atom inthe cyclic moiety, e.g., a nitrogenatom) or an exocyclic atom (i.e., anatom or group of atoms attached to a constituent atom in the cyclicmoiety).

The carrier can be any organic molecule containing attachment points forOFG¹, OFG², and the ligand. In certain embodiments, carrier is a cyclicmolecule and may contain heteroatoms (e.g., O, N or S). E.g., carriermolecules may include aryl (e.g., benzene, biphenyl, etc.), cycloalkyl(e.g., cyclohexane, cis or trans decalin, etc.), or heterocyclyl(piperazine, pyrrolidine, etc.). In other embodiments, the carrier canbe an acyclic moiety, e.g., based on serinol. Any of the above cyclicsystems may include substituents in addition to OFG¹, OFG², and theligand.

Sugar-Based Monomers

In some embodiments, the carrier molecule is an oxygen containingheterocycle. Preferably the carrier is a ribose sugar as shown instructure LCM-I. In this embodiment, the ligand-conjugated monomer is anucleoside.

“B” represents a nucleobase, e.g., a naturally occurring nucleobaseoptionally having one or more chemical modifications, e.g., and unusualbase; or a universal base.

As used herein, an “unusual” nucleobase can include any one of thefollowing:

-   2-methyladeninyl,-   N6-methyladeninyl,-   2-methylthio-N-6-methyladeninyl,-   N6-isopentenyladeninyl,-   2-methylthio-N-6-isopentenyladeninyl,-   N6-(cis-hydroxyisopentenyl)adeninyl,-   2-methylthio-N-6-(cis-hydroxyisopentenyl)adeninyl,-   N6-glycinylcarbamoyladeninyl,-   N6-threonylcarbamoyladeninyl,-   2-methylthio-N-6-threonyl carbamoyladeninyl,-   N6-methyl-N-6-threonylcarbamoyladeninyl,-   N6-hydroxynorvalylcarbamoyladeninyl,-   2-methylthio-N-6-hydroxynorvalyl carbamoyladeninyl,-   N6,N6-dimethyladeninyl,-   3-methylcytosinyl,-   5-methylcytosinyl,-   2-thiocytosinyl,-   5-formylcytosinyl,

-   N4-methylcytosinyl,-   5-hydroxymethylcytosinyl,-   1-methylguaninyl,-   N2-methylguaninyl,-   7-methylguaninyl,-   N2,N2-dimethylguaninyl,

-   N2,7-dimethylguaninyl,-   N2,N2,7-trimethylguaninyl,-   1-methylguaninyl,-   7-cyano-7-deazaguaninyl,-   7-aminomethyl-7-deazaguaninyl,-   pseudouracilyl,-   dihydrouracilyl,-   5-methyluracilyl,-   1-methylpseudouracilyl,-   2-thiouracilyl,-   4-thiouracilyl,-   2-thiothyminyl-   5-methyl-2-thiouracilyl,-   3-(3-amino-3-carboxypropyl)uracilyl,-   5-hydroxyuracilyl,-   5-methoxyuracilyl,-   uracilyl 5-oxyacetic acid,-   uracilyl 5-oxyacetic acid methyl ester,-   5-(carboxyhydroxymethyl)uracilyl,-   5-(carboxyhydroxymethyl)uracilyl methyl ester,-   5-methoxycarbonylmethyluracilyl,-   5-methoxycarbonylmethyl-2-thiouracilyl,-   5-aminomethyl-2-thiouracilyl,-   5-methylaminomethyluracilyl,-   5-methylaminomethyl-2-thiouracilyl,-   5-methylaminomethyl-2-selenouracilyl,-   5-carbamoylmethyluracilyl, 5-carboxymethylaminomethyluracilyl,-   5-carboxymethylaminomethyl-2-thiouracilyl,-   3-methyluracilyl,-   1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,-   5-carboxymethyluracilyl,-   5-methyldihydrouracilyl, or-   3-methylpseudouracilyl.

A universal base can form base pairs with each of the natural DNA/RNAbases, exhibiting relatively little discrimination between them. Ingeneral, the universal bases are non-hydrogen bonding, hydrophobic,aromatic moieties which can stabilize e.g., duplex RNA or RNA-likemolecules, via stacking interactions. A universal base can also includehydrogen bonding substituents.

As used herein, a “universal base” can include anthracenes, pyrenes orany one of the following:

In some embodiments, B can form part of a tether that connects a ligandto the carrier. For example, the tether can beB—CH═CH—C(O)NH—(CH₂)₅—NHC(O)-LIGAND. In a preferred embodiment, thedouble bond is trans, and the ligand is a substituted or unsubstitutedcholesterolyl radical (e.g., attached through the D-ring side chain orthe C-3 hydroxyl); an aralkyl moiety having at least one sterogeniccenter and at least one substituent on the aryl portion of the aralkylgroup; or a nucleobase. In certain embodiments, B, in the tetherdescribed above, is uracilyl or a universal base, e.g., an aryl moiety,e.g., phenyl, optionally having additional substituents, e.g., one ormore fluoro groups. B can be substituted at any atom with the remainderof the tether.

X² can include “oxy” or “deoxy” substituents in place of the 2′-OH or bea ligand or a tethered ligand.

Examples of “oxy”-substituents include alkoxy or aryloxy (OR, e.g., R═H,alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, sugar, or protectinggroup); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O—PROTECTEDAMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino,diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)PROTECTED AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino),and orthoester. Amine protecting groups can include formyl, amido,benzyl, allyl, etc.

Preferred orthoesters have the general formula J. The groups R³¹ and R³²may be the same or different. A preferred orthoester is the “ACE” group,shown below as structure K.

1.

“Deoxy” substituents include hydrogen (i.e. deoxyribose sugars); halo(e.g., fluoro); protected amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid in which all amino are protected); fully protectedpolyamino (e.g., NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE, wherein AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino and all amino groups areprotected), —NHC(O)R(R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar), cyano; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl,aryl, alkenyl and alkynyl, which may be optionally substituted withe.g., a protected amino functionality. Preferred substitutents are2′-methoxyethyl, 2′-OCH3,2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

X³ is as described for OFG² above.

PG can be a triarylmethyl group (e.g., a dimethoxytrityl group) orSi(X^(5′))(X^(5″))(X^(5′″)) in which (X^(5′)), (X^(5″)), and (X^(5′″))are as described elsewhere.

Sugar Replacement-Based Monomers

Cyclic sugar replacement-based monomers, e.g., sugar replacement-basedligand-conjugated monomers, are also referred to herein as sugarreplacement monomer subunit (SRMS) monomer compounds. Preferred carriershave the general formula (LCM-2) provided below. (In that structurepreferred backbone attachment points can be chosen from R¹ or R²; R³(two positions are chosen to give two backbone attachment points, e.g.,R¹ and R⁴, or R⁴ and R⁹). Preferred tethering attachment points includeR⁷; R⁵ or R⁶ when X is CH₂. The carriers are described below as anentity, which can be incorporated into a strand. Thus, it is understoodthat the structures also encompass the situations wherein one (in thecase of a terminal position) or two (in the case of an internalposition) of the attachment points, e.g., R¹ or R²; R³ or R⁴; or R⁹ orR¹⁰ (when Y is CR⁹R¹⁰), is connected to the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone. E.g., one of theabove-named R groups can be —CH₂—, wherein one bond is connected to thecarrier and one to a backbone atom, e.g., a linking oxygen or a centralphosphorus atom.

in which,

X is N(CO)R⁷, NR⁷ or CH₂;

Y is NR⁸, O, S, CR⁹R¹⁰;

Z is CR¹¹R¹² or absent;

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a), or(CH₂)_(n)OR^(b), provided that at least two of R¹, R², R³, R⁴, R⁹, andR¹⁰ are OR^(a) and/or (CH₂)_(n)OR^(b);

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, a ligand, H, C₁-C₆ alkyloptionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ togetherare C₃-C₈ cycloalkyl optionally substituted with R¹⁴;

R⁷ can be a ligand, e.g., R⁷ can be R^(d), or R⁷ can be a ligandtethered indirectly to the carrier, e.g., through a tethering moiety,e.g., C₁-C₂₀ alkyl substituted with NR^(c)R^(d); or C₁-C₂₀ alkylsubstituted with NHC(O)R^(d);

R⁸ is H or C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR^(c)R⁷;

R¹⁵ is C₁-C₆ alkyl optionally substituted with cyano, or C₂-C₆ alkenyl;

R¹⁶ is C₁-C₁₀ alkyl;

R¹⁷ is a liquid or solid phase support reagent;

L is —C(O)(CH₂)_(q)C(O)—, or —C(O)(CH₂)_(g)S—;

R^(a) is a protecting group, e.g., CAr₃; (e.g., a dimethoxytrityl group)or Si(X^(5′))(X^(5″))(X^(5′″)) in which (X^(5′)), (X^(5″)), and(X^(5′″)) are as described elsewhere.

R^(b) is P(O)(O⁻)H, P(OR¹⁵)N(R¹⁶)₂ or L-R¹⁷;

R^(c) is H or C₁-C₆ alkyl;

R^(d) is H or a ligand;

Each Ar is, independently, C₆-C₁₀ aryl optionally substituted with C₁-C₄alkoxy;

n is 1-4; and q is 0-4.

Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Yis CR⁹R¹⁰, and or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹²; or Xis N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹²; or X is CH₂; Y is CR⁹R¹⁰; Zis CR¹¹R¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or theindane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵and R¹¹ together form C₅ cycloalkyl (H, z=1).

In certain embodiments, the carrier may be based on the pyrroline ringsystem or the 4-hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Yis CR⁹R¹⁰, and Z is absent (D). OFG¹ is preferably attached to a primarycarbon, e.g., an exocyclic alkylene

group, e.g., a methylene group, connected to one of the carbons in thefive-membered ring (—CH₂OFG¹ in D). OFG² is preferably attached directlyto one of the carbons in the five-membered ring (—OFG² in D). For thepyrroline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² maybe attached to C-3; or —CH₂OFG¹ may be attached to C-3 and OFG² may beattached to C-4. In certain embodiments, CH₂OFG¹ and OFG² may begeminally substituted to one of the above-referenced carbons. For the3-hydroxyproline-based carriers, —CH₂OFG¹ may be attached to C-2 andOFG² may be attached to C-4. The pyrroline- and 4-hydroxyproline-basedmonomers may therefore contain linkages (e.g., carbon-carbon bonds)wherein bond rotation is restricted about that particular linkage, e.g.restriction resulting from the presence of a ring. Thus, CH₂OFG¹ andOFG² may be cis or trans with respect to one another in any of thepairings delineated above Accordingly, all cis/trans isomers areexpressly included. The monomers may also contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of the monomers are expressly included (e.g., thecenters bearing CH₂OFG¹ and OFG² can both have the R configuration; orboth have the S configuration; or one center can have the Rconfiguration and the other center can have the S configuration and viceversa). The tethering attachment point is preferably nitrogen. Preferredexamples of carrier D include the following:

In certain embodiments, the carrier may be based on the piperidine ringsystem (E), e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., amethylene group (n=1) or ethylene group (n=2), connected to one of thecarbons in the six-membered ring [—(CH₂)_(n)OFG¹ in E]. OFG² ispreferably attached directly to one of the carbons in the six-memberedring (—OFG² in E). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminalmanner on the ring, i.e., both groups may be attached to the samecarbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH₂)_(n)OFG¹ andOFG² may be disposed in a vicinal manner on the ring, i.e., both groupsmay be attached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ maybe attached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ maybe attached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ maybe attached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹may be attached to C-4 and OFG² may be attached to C-3. Thepiperidine-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect toone another in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). The tethering attachment point ispreferably nitrogen.

In certain embodiments, the carrier may be based on the piperazine ringsystem (F), e.g., X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹², orthe morpholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Zis CR¹¹R¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., amethylene group, connected to one of the carbons in the six-memberedring (—CH₂OFG¹ in F or G). OFG² is preferably attached directly to oneof the carbons in the six-membered rings (—OFG² in F or G). For both Fand G, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3;or vice versa. In certain embodiments, CH₂OFG¹ and OFG² may be geminallysubstituted to one of the above-referenced carbons. The piperazine- andmorpholine-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to oneanother in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). R′″ can be, e.g., C₁-C₆ alkyl, preferablyCH₃. The tethering attachment point is preferably nitrogen in both F andG.

In certain embodiments, the carrier may be based on the decalin ringsystem, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., Xis CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₅cycloalkyl (H, z=1). OFG¹ is preferably attached to a primary carbon,

e.g., an exocyclic methylene group (n=1) or ethylene group (n=2)connected to one of C-2, C-3, C-4, or C-5 [—(CH₂)_(n)OFG¹ in H]. OFG² ispreferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG² inH). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminal manner on thering, i.e., both groups may be attached to the same carbon, e.g., atC-2, C-3, C-4, or C-5. Alternatively, —(CH₂)_(n)OFG¹ and OFG² may bedisposed in a vicinal manner on the ring, i.e., both groups may beattached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ may beattached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may beattached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ may beattached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹ maybe attached to C-4 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ maybe attached to C-4 and OFG² may be attached to C-5; or —(CH₂)_(n)OFG¹may be attached to C-5 and OFG² may be attached to C-4. The decalin orindane-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect toone another in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). In a preferred embodiment, thesubstituents at C-1 and C-6 are trans with respect to one another. Thetethering attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus,—(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another.Accordingly, all cis/trans isomers are expressly included. The monomersmay also contain one or more asymmetric centers

and thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of the monomers are expressly included (e.g., the centers bearingCH₂OFG¹ and OFG² can both have the R configuration; or both have the Sconfiguration; or one center can have the R configuration and the othercenter can have the S configuration and vice versa). The tetheringattachment point is preferably nitrogen.

Sugar Replacement-Based Monomers (Acyclic)

Acyclic sugar replacement-based monomers, e.g., sugar replacement-basedligand-conjugated monomers, are also referred to herein as sugarreplacement monomer subunit (SRMS) monomer compounds. Preferred acycliccarriers can have formula LCM-3 or LCM-4 below.

In some embodiments, each of x, y, and z can be, independently of oneanother, 0, 1, 2, or 3. In formula LCM-3, when y and z are different,then the tertiary carbon can have either the R or S configuration. Inpreferred embodiments, x is zero and y and z are each 1 in formulaLCM-3(e.g., based on serinol), and y and z are each 1 in formula LCM-3.Each of formula LCM-3 or LCM-4 below can optionally be substituted,e.g., with hydroxy, alkoxy, perhaloalkyl.

Tethers

In certain embodiments, a moiety, e.g., a ligand may be connectedindirectly to the carrier via the intermediacy of an intervening tether.Tethers are connected to the carrier at a tethering attachment point(TAP) and may include any C₁-C₁₀₀ carbon-containing moiety, (e.g.C₁-C₇₅, C₁-C₅₀, C₁-C₂₀, C₁-C₁₀; C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, orC₁₀), preferably having at least one nitrogen atom. In preferredembodiments, the nitrogen atom forms part of a terminal amino or amido(NHC(O)—) group on the tether, which may serve as a connection point forthe ligand. Preferred tethers (underlined) include TAP-(CH₂)_(n)NH—;TAP-C(O)(CH₂)_(n)NH; TAP-NR′″″(CH₂)_(n)NH—, TAP-C(O)—(CH₂)_(n)—C(O)—;TAP-C(O)—(CH₂)_(n)—C(O)O—; TAP-C(O)—O—; TAP-C(O)—(CH₂)_(n)—NH—C(O)—;TAP-C(O)—(CH₂)_(n)—; TAP-C(O)—NH—; TAP-C(O)—; TAP-(CH₂)_(n)—C(O)—;TAP-(CH₂)_(n)—C(O)O—; TAP-(CH₂)_(n) ; or TAP-(CH₂)_(n)—NH—C(O)—; inwhich n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20) and R″″ is C₁-C₆ alkyl. Preferably, n is 5,6, or 11. In other embodiments, the nitrogen may form part of a terminaloxyamino group, e.g., —ONH₂, or hydrazino group, —NHNH₂. The tether mayoptionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl,and/or optionally inserted with one or more additional heteroatoms,e.g., N, O, or S. Preferred tethered ligands may include, e.g.,TAP-(CH₂)_(n)NH(LIGAND); TAP-C(O)(CH₂)_(n)NH(LIGAND);TAP-NR″″(CH₂)_(n)NH(LIGAND); TAP-(CH₂)_(n)ONH(LIGAND);TAP-C(O)(CH₂)_(n)ONH(LIGAND); TAP-NR″″(CH₂)_(n)ONH(LIGAND);TAP-(CH₂)_(n)NHNH₂(LIGAND), TAP-C(O)(CH₂)_(n)NHNH₂(LIGAND);TAP-NR″″(CH₂)_(n)NHNH₂(LIGAND); TAP-C(O)—(CH₂)_(n)—C(O)(LIGAND);TAP-C(O)—(CH₂)_(n)—C(O)O(LIGAND); TAP-C(O)—O(LIGAND);TAP-C(O)—(CH₂)_(n)—NH—C(O)(LIGAND); TAP-C(O)—(CH₂)_(n)(LIGAND);TAP-C(O)—NH(LIGAND); TAP-C(O)(LIGAND); TAP-(CH₂)_(n)—C(O) (LIGAND);TAP-(CH₂)_(n)—C(O)O(LIGAND); TAP-(CH₂)_(n)(LIGAND); orTAP-(CH₂)_(n)—NH—C(O)(LIGAND). In some embodiments, amino terminatedtethers (e.g., NH₂, ONH₂, NH₂NH₂) can form an imino bond (i.e., C═N)with the ligand. In some embodiments, amino terminated tethers (e.g.,NH₂, ONH₂, NH₂NH₂) can acylated, e.g., with C(O)CF₃.

In some embodiments, the tether can terminate with a mercapto group(i.e., SH) or an olefin (e.g., CH═CH₂). For example, the tether can beTAP-(CH₂)_(n)—SH, TAP-C(O)(CH₂)_(n)SH, TAP-(CH₂)_(n)—(CH═CH₂), orTAP-C(O)(CH₂)_(n)(CH═CH₂), in which n can be as described elsewhere. Incertain embodiments, the olefin can be a Diels-Alder diene ordienophile. The tether may optionally be substituted, e.g., withhydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one ormore additional heteroatoms, e.g., N, O, or S. The double bond can becis or trans or E or Z.

In other embodiments the tether may include an electrophilic moiety,preferably at the terminal position of the tether. Preferredelectrophilic moieties include, e.g., an aldehyde, alkyl halide,mesylate, tosylate, nosylate, or brosylate, or an activated carboxylicacid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferredtethers (underlined) include TAP-(CH₂)_(n)CHO; TAP-C(O)(CH₂)_(n)CHO; orTAP-NR″″(CH₂)_(n)CHO, in which n is 1-6 and R″″ is C₁-C₆ alkyl; orTAP-(CH₂)_(n)C(O)ONHS; TAP-C(O)(CH₂)_(n)C(O)ONHS; orTAP-NR″″(CH₂)_(n)C(O)ONHS, in which n is 1-6 and R″″ is C₁-C₆ alkyl;TAP-(CH₂)_(n)C(O)OC₆F₅ ; TAP-C(O)(CH₂)_(n)C(O)OC₆F₅ ; orTAP-NR″″(CH₂)_(n)C(O)CO₆F₅ , in which n is 1-11 and R″″ is C₁-C₆ alkyl;or —(CH₂)_(n)CH₂LG; TAP-C(O)(CH₂)_(n)CH₂LG; or TAP-NR″″(CH₂)_(n)CH₂LG,in which n can be as described elsewhere and R″″ is C₁-C₆ alkyl (LG canbe a leaving group, e.g., halide, mesylate, tosylate, nosylate,brosylate). Tethering can be carried out by coupling a nucleophilicgroup of a ligand, e.g., a thiol or amino group with an electrophilicgroup on the tether.

In other embodiments, it can be desirable for the ligand-conjugatedmonomer or a ligand-conjugated monomer to include a phthalimido group(K) at the terminal position of the tether.

In other embodiments, other protected amino groups can be at theterminal position of the tether, e.g., alloc, monomethoxy trityl (MMT),trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can beortho-nitrophenyl or ortho, para-dinitrophenyl).

Any of the tethers described herein may further include one or moreadditional linking groups, e.g., —O—(CH₂)_(n)—, —(CH₂)_(n)—SS—,—(CH₂)_(n)—, or —(CH═CH)—.

Asymmetrical Modifications

An RNA, e.g., an iRNA agent, can be asymmetrically modified as describedherein, and as described in International Application Serial No.PCT/US04/07070, filed Mar. 8, 2004, which is hereby incorporated byreference.

An asymmetrically modified iRNA agent is one in which a strand has amodification which is not present on the other strand. An asymmetricalmodification is a modification found on one strand but not on the otherstrand. Any modification, e.g., any modification described herein, canbe present as an asymmetrical modification. An asymmetrical modificationcan confer any of the desired properties associated with a modification,e.g., those properties discussed herein. E.g., an asymmetricalmodification can: confer resistance to degradation, an alteration inhalf life; target the iRNA agent to a particular target, e.g., to aparticular tissue; modulate, e.g., increase or decrease, the affinity ofa strand for its complement or target sequence; or hinder or promotemodification of a terminal moiety, e.g., modification by a kinase orother enzymes involved in the RISC mechanism pathway. The designation ofa modification as having one property does not mean that it has no otherproperty, e.g., a modification referred to as one which promotesstabilization might also enhance targeting.

While not wishing to be bound by theory or any particular mechanisticmodel, it is believed that asymmetrical modification allows an iRNAagent to be optimized in view of the different or “asymmetrical”functions of the sense and antisense strands. For example, both strandscan be modified to increase nuclease resistance, however, since somechanges can inhibit RISC activity, these changes can be chosen for thesense stand. In addition, since some modifications, e.g., targetingmoieties, can add large bulky groups that, e.g., can interfere with thecleavage activity of the RISC complex, such modifications are preferablyplaced on the sense strand. Thus, targeting moieties, especially bulkyones (e.g. cholesterol), are preferentially added to the sense strand.In one embodiment, an asymmetrical modification in which a phosphate ofthe backbone is substituted with S, e.g., a phosphorothioatemodification, is present in the antisense strand, and a 2′ modification,e.g., 2′ OMe is present in the sense strand. A targeting moiety can bepresent at either (or both) the 5′ or 3′ end of the sense strand of theiRNA agent. In a preferred example, a P of the backbone is replaced withS in the antisense strand, 2′OMe is present in the sense strand, and atargeting moiety is added to either the 5′ or 3′ end of the sense strandof the iRNA agent.

In a preferred embodiment an asymmetrically modified iRNA agent has amodification on the sense strand which modification is not found on theantisense strand and the antisense strand has a modification which isnot found on the sense strand.

Each strand can include one or more asymmetrical modifications. By wayof example: one strand can include a first asymmetrical modificationwhich confers a first property on the iRNA agent and the other strandcan have a second asymmetrical modification which confers a secondproperty on the iRNA. E.g., one strand, e.g., the sense strand can havea modification which targets the iRNA agent to a tissue, and the otherstrand, e.g., the antisense strand, has a modification which promoteshybridization with the target gene sequence.

In some embodiments both strands can be modified to optimize the sameproperty, e.g., to increase resistance to nucleolytic degradation, butdifferent modifications are chosen for the sense and the antisensestrands, e.g., because the modifications affect other properties aswell. E.g., since some changes can affect RISC activity thesemodifications are chosen for the sense strand.

In one embodiment, one strand has an asymmetrical 2′ modification, e.g.,a 2′ OMe modification, and the other strand has an asymmetricalmodification of the phosphate backbone, e.g., a phosphorothioatemodification. So, in one embodiment the antisense strand has anasymmetrical 2′ OMe modification and the sense strand has anasymmetrical phosphorothioate modification (or vice versa). In aparticularly preferred embodiment, the RNAi agent will have asymmetrical2′-O alkyl, preferably, 2′-OMe modifications on the sense strand andasymmetrical backbone P modification, preferably a phosphorothioatemodification in the antisense strand. There can be one or multiple2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunitsof the sense strand can be so modified. There can be one or multiplephosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of thesubunits of the antisense strand can be so modified. It is preferable tohave an iRNA agent wherein there are multiple 2′-OMe modifications onthe sense strand and multiple phosphorothioate modifications on theantisense strand. All of the subunits on one or both strands can be somodified. A particularly preferred embodiment of multiple asymmetricmodifications on both strands has a duplex region about 20-21, andpreferably 19, subunits in length and one or two 3′ overhangs of about 2subunits in length.

Asymmetrical modifications are useful for promoting resistance todegradation by nucleases, e.g., endonucleases. iRNA agents can includeone or more asymmetrical modifications which promote resistance todegradation. In preferred embodiments the modification on the antisensestrand is one which will not interfere with silencing of the target,e.g., one which will not interfere with cleavage of the target. Most ifnot all sites on a strand are vulnerable, to some degree, to degradationby endonucleases. One can determine sites which are relativelyvulnerable and insert asymmetrical modifications which inhibitdegradation. It is often desirable to provide asymmetrical modificationof a UA site in an iRNA agent, and in some cases it is desirable toprovide the UA sequence on both strands with asymmetrical modification.Examples of modifications which inhibit endonucleolytic degradation canbe found herein. Particularly favored modifications include: 2′modification, e.g., provision of a 2′ OMe moiety on the U, especially ona sense strand; modification of the backbone, e.g., with the replacementof an 0 with an S, in the phosphate backbone, e.g., the provision of aphosphorothioate modification, on the U or the A or both, especially onan antisense strand; replacement of the U with a C5 amino linker;replacement of the A with a G (sequence changes are preferred to belocated on the sense strand and not the antisense strand); andmodification of the at the 2′, 6′, 7′, or 8′ position. Preferredembodiments are those in which one or more of these modifications arepresent on the sense but not the antisense strand, or embodiments wherethe antisense strand has fewer of such modifications.

Asymmetrical modification can be used to inhibit degradation byexonucleases. Asymmetrical modifications can include those in which onlyone strand is modified as well as those in which both are modified. Inpreferred embodiments the modification on the antisense strand is onewhich will not interfere with silencing of the target, e.g., one whichwill not interfere with cleavage of the target. Some embodiments willhave an asymmetrical modification on the sense strand, e.g., in a 3′overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g.,in a 3′ overhang, e.g., at the 3′ terminus. If the modificationsintroduce moieties of different size it is preferable that the larger beon the sense strand. If the modifications introduce moieties ofdifferent charge it is preferable that the one with greater charge be onthe sense strand.

Examples of modifications which inhibit exonucleolytic degradation canbe found herein. Particularly favored modifications include: 2′modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g.,at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule orat the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicatedby the context); modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P in a 3′ overhang, e.g., atthe 3′ terminus; combination of a 2′ modification, e.g., provision of a2′O Me moiety and modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P, in a 3′ overhang, e.g., atthe 3′ terminus; modification with a 3′ alkyl; modification with anabasic pyrrolidine in a 3′ overhang, e.g., at the 3′ terminus;modification with naproxene, ibuprofen, or other moieties which inhibitdegradation at the 3′ terminus. Preferred embodiments are those in whichone or more of these modifications are present on the sense but not theantisense strand, or embodiments where the antisense strand has fewer ofsuch modifications.

Modifications, e.g., those described herein, which affect targeting canbe provided as asymmetrical modifications. Targeting modifications whichcan inhibit silencing, e.g., by inhibiting cleavage of a target, can beprovided as asymmetrical modifications of the sense strand. Abiodistribution altering moiety, e.g., cholesterol, can be provided inone or more, e.g., two, asymmetrical modifications of the sense strand.Targeting modifications which introduce moieties having a relativelylarge molecular weight, e.g., a molecular weight of more than 400, 500,or 1000 daltons, or which introduce a charged moiety (e.g., having morethan one positive charge or one negative charge) can be placed on thesense strand.

Modifications, e.g., those described herein, which modulate, e.g.,increase or decrease, the affinity of a strand for its compliment ortarget, can be provided as asymmetrical modifications. These include: 5methyl U; 5 methyl C; pseudouridine, Locked nucleic acids include: 2thio U and 2-amino-A. In some embodiments one or more of these isprovided on the antisense strand.

iRNA agents have a defined structure, with a sense strand and anantisense strand, and in many cases short single strand overhangs, e.g.,of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetricalmodification can be used to optimize the activity of such a structure,e.g., by being placed selectively within the iRNA. E.g., the end regionof the iRNA agent defined by the 5′ end of the sense strand and the 3′end of the antisense strand is important for function. This region caninclude the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang.In preferred embodiments asymmetrical modifications which result in oneor more of the following are used: modifications of the 5′ end of thesense strand which inhibit kinase activation of the sense strand,including, e.g., attachments of conjugates which target the molecule orthe use modifications which protect against 5′ exonucleolyticdegradation; or modifications of either strand, but preferably the sensestrand, which enhance binding between the sense and antisense strand andthereby promote a “tight” structure at this end of the molecule.

The end region of the iRNA agent defined by the 3′ end of the sensestrand and the 5′ end of the antisense strand is also important forfunction. This region can include the terminal 2, 3, or 4 pairednucleotides and any 3′ overhang. Preferred embodiments includeasymmetrical modifications of either strand, but preferably the sensestrand, which decrease binding between the sense and antisense strandand thereby promote an “open” structure at this end of the molecule.Such modifications include placing conjugates which target the moleculeor modifications which promote nuclease resistance on the sense strandin this region. Modification of the antisense strand which inhibitkinase activation are avoided in preferred embodiments.

Exemplary modifications for asymmetrical placement in the sense strandinclude the following:

(a) backbone modifications, e.g., modification of a backbone P,including replacement of P with S, or P substituted with alkyl or allyl,e.g., Me, and dithioates (S—P═S); these modifications can be used topromote nuclease resistance;

(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminaland/or internal positions); these modifications can be used to promotenuclease resistance or to enhance binding of the sense to the antisensestrand, the 3′ modifications can be used at the 5′ end of the sensestrand to avoid sense strand activation by RISC;

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S)these modifications can be used to promote nuclease resistance or toinhibit binding of the sense to the antisense strand, or can be used atthe 5′ end of the sense strand to avoid sense strand activation by RISC;

(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe);these modifications can be used to promote nuclease resistance or toinhibit binding of the sense to the antisense strand, or can be used atthe 5′ end of the sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleicacids (HNA's) and cyclohexene nucleic acids (CeNA's)); thesemodifications can be used to promote nuclease resistance or to inhibitbinding of the sense to the antisense strand, or can be used at the 5′end of the sense strand to avoid sense strand activation by RISC;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2modified purines, N-7 modified purines, N-6 modified purines), thesemodifications can be used to promote nuclease resistance or to enhancebinding of the sense to the antisense strand;

(g) cationic groups and Zwitterionic groups (preferably at a terminus),these modifications can be used to promote nuclease resistance;

(h) conjugate groups (preferably at terminal positions), e.g., naproxen,biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates;these modifications can be used to promote nuclease resistance or totarget the molecule, or can be used at the 5′ end of the sense strand toavoid sense strand activation by RISC.

Exemplary modifications for asymmetrical placement in the antisensestrand include the following:

(a) backbone modifications, e.g., modification of a backbone P,including replacement of P with S, or P substituted with alkyl or allyl,e.g., Me, and dithioates (S—P═S);

(b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the3′ end); e.g., with P═O or P═S preferably at the 3′-end, thesemodifications are preferably excluded from the 5′ end region as they mayinterfere with RISC enzyme activity such as kinase activity;

(d) L sugars (e.g, L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe);e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the3′-end, these modifications are preferably excluded from the 5′ endregion as they may interfere with kinase activity;

(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modificationsare preferably excluded from the 5′ end region as they may contribute tounwanted enhancements of paring between the sense and antisense strands,it is often preferred to have a “loose” structure in the 5′ region,additionally, they may interfere with kinase activity;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2modified purines, N-7 modified purines, N-6 modified purines);

(g) cationic groups and Zwitterionic groups (preferably at a terminus);

cationic groups and Zwitterionic groups at 2′-position of sugar;3′-position of the sugar; as nucleobase modifications (e.g., C-5modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6modified purines);

conjugate groups (preferably at terminal positions), e.g., naproxen,biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates,but bulky groups or generally groups which inhibit RISC activity shouldare less preferred.

The 5′-OH of the antisense strand should be kept free to promoteactivity. In some preferred embodiments modifications that promotenuclease resistance should be included at the 3′ end, particularly inthe 3′ overhang.

In another aspect, the invention features a method of optimizing, e.g.,stabilizing, an iRNA agent. The method includes selecting a sequencehaving activity, introducing one or more asymmetric modifications intothe sequence, wherein the introduction of the asymmetric modificationoptimizes a property of the iRNA agent but does not result in a decreasein activity.

The decrease in activity can be less than a preselected level ofdecrease. In preferred embodiments decrease in activity means a decreaseof less than 5, 10, 20, 40, or 50% activity, as compared with anotherwise similar iRNA lacking the introduced modification. Activitycan, e.g., be measured in vivo, or in vitro, with a result in eitherbeing sufficient to demonstrate the required maintenance of activity.

The optimized property can be any property described herein and inparticular the properties discussed in the section on asymmetricalmodifications provided herein. The modification can be any asymmetricalmodification, e.g., an asymmetric modification described in the sectionon asymmetrical modifications described herein. Particularly preferredasymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMemodifications, particularly in the sense sequence, and modifications ofa backbone 0, particularly phosphorothioate modifications, in theantisense sequence.

In a preferred embodiment a sense sequence is selected and provided withan asymmetrical modification, while in other embodiments an antisensesequence is selected and provided with an asymmetrical modification. Insome embodiments both sense and antisense sequences are selected andeach provided with one or more asymmetrical modifications.

Multiple asymmetric modifications can be introduced into either or bothof the sense and antisense sequence. A sequence can have at least 2, 4,6, 8, or more modifications and all or substantially all of the monomersof a sequence can be modified.

Differential Modification of Terminal Duplex Stability

In one aspect, the invention features an iRNA agent which can havedifferential modification of terminal duplex stability (DMTDS).

In addition, the invention includes iRNA agents having DMTDS and anotherelement described herein. E.g., the invention includes an iRNA agentdescribed herein, e.g., a palindromic iRNA agent, an iRNA agent having anon canonical pairing, an iRNA agent which targets a gene describedherein, e.g., an htt gene, an iRNA agent having an architecture orstructure described herein, an iRNA associated with an amphipathicdelivery agent described herein, an iRNA associated with a drug deliverymodule described herein, an iRNA agent administered as described herein,or an iRNA agent formulated as described herein, which also incorporatesDMTDS.

iRNA agents can be optimized by increasing the propensity of the duplexto disassociate or melt (decreasing the free energy of duplexassociation), in the region of the 5′ end of the antisense strandduplex. This can be accomplished, e.g., by the inclusion of subunits,which increase the propensity of the duplex to disassociate or melt inthe region of the 5′ end of the antisense strand. This can also beaccomplished by the attachment of a ligand that increases the propensityof the duplex to disassociate of melt in the region of the 5′ end. Whilenot wishing to be bound by theory, the effect may be due to promotingthe effect of an enzyme such as a helicase, for example, promoting theeffect of the enzyme in the proximity of the 5′ end of the antisensestrand.

The inventors have also discovered that iRNA agents can be optimized bydecreasing the propensity of the duplex to disassociate or melt(increasing the free energy of duplex association), in the region of the3′ end of the antisense strand duplex. This can be accomplished, e.g.,by the inclusion of subunits which decrease the propensity of the duplexto disassociate or melt in the region of the 3′ end of the antisensestrand. It can also be accomplished by the attachment of ligand thatdecreases the propensity of the duplex to disassociate or melt in theregion of the 5′ end.

Modifications which increase the tendency of the 5′ end of the duplex todissociate can be used alone or in combination with other modificationsdescribed herein, e.g., with modifications which decrease the tendencyof the 3′ end of the duplex to dissociate. Likewise, modifications whichdecrease the tendency of the 3′ end of the duplex to dissociate can beused alone or in combination with other modifications described herein,e.g., with modifications which increase the tendency of the 5′ end ofthe duplex to dissociate.

Decreasing the Stability of the AS 5′ end of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promotedissociation or melting (e.g., on the free energy of association ordissociation of a particular pairing, the simplest approach is toexamine the pairs on an individual pair basis, though next neighbor orsimilar analysis can also be used). In terms of promoting dissociation:

A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferredover G:C (I = inosine);

-   -   mismatches, e.g., non-canonical or other than canonical pairings        (as described elsewhere herein) are preferred over canonical        (A:T, A:U, G:C) pairings;    -   pairings which include a universal base are preferred over        canonical pairings.

A typical ds iRNA agent can be diagrammed as follows:

S 5′ R₁ N₁ N₂ N₃ N₄ N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₂ 3′ AS 3′ R₃ N₁ N₂ N₃N₄ N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₄ 5′ S:AS P₁ P₂ P₃ P₄ P₅ [N] P⁻⁵ P⁻⁴ P⁻³P⁻² P⁻¹ 5′

S indicates the sense strand; AS indicates antisense strand; R₁indicates an optional (and nonpreferred) 5′ sense strand overhang; R₂indicates an optional (though preferred) 3′ sense overhang; R₃ indicatesan optional (though preferred) 3′ antisense sense overhang; R₄ indicatesan optional (and nonpreferred) 5′ antisense overhang; N indicatessubunits; [N] indicates that additional subunit pairs may be present;and P_(x), indicates a paring of sense N_(x) and antisense N_(x).Overhangs are not shown in the P diagram. In some embodiments a 3′ ASoverhang corresponds to region Z, the duplex region corresponds toregion X, and the 3′ S strand overhang corresponds to region Y, asdescribed elsewhere herein. (The diagram is not meant to imply maximumor minimum lengths, on which guidance is provided elsewhere herein.)

It is preferred that pairings which decrease the propensity to form aduplex are used at 1 or more of the positions in the duplex at the 5′end of the AS strand. The terminal pair (the most 5′ pair in terms ofthe AS strand) is designated as P⁻¹, and the subsequent pairingpositions (going in the 3′ direction in terms of the AS strand) in theduplex are designated, P⁻², P⁻³, P⁻⁴, P⁻⁵, and so on. The preferredregion in which to modify or modulate duplex formation is at P⁻⁵ throughP⁻¹, more preferably P⁻⁴ through P⁻¹, more preferably P⁻³ through P⁻¹.Modification at P⁻¹, is particularly preferred, alone or withmodification(s) other position(s), e.g., any of the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions be chosenindependently from the group of:

-   -   A:U    -   G:U    -   I:C    -   mismatched pairs, e.g., non-canonical or other than canonical        pairings or pairings which include a universal base.

In preferred embodiments the change in subunit needed to achieve apairing which promotes dissociation will be made in the sense strand,though in some embodiments the change will be made in the antisensestrand.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairs which promote dissociation.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are G:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are I:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are mismatched pairs, e.g., non-canonical or other thancanonical pairings pairings.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairings which include a universal base.

Increasing the Stability of the AS 3′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promotestability and inhibit dissociation or melting (e.g., on the free energyof association or dissociation of a particular pairing, the simplestapproach is to examine the pairs on an individual pair basis, thoughnext neighbor or similar analysis can also be used). In terms ofpromoting duplex stability:

G:C is preferred over A:U

-   -   Watson-Crick matches (A:T, A:U, G:C) are preferred over        non-canonical or other than canonical pairings    -   analogs that increase stability are preferred over Watson-Crick        matches (A:T, A:U, G:C)

2-amino-A:U is preferred over A:U 2-thio U or 5 Me-thio-U:A arepreferred over U:A G-clamp is preferred over C:G (an analog of C having4 hydrogen bonds):G guanadinium-G-clamp:G is preferred over C:G pseudouridine:A is preferred over U:A

-   -   sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or        LNA, which enhance binding are preferred over non-modified        moieties and can be present on one or both strands to enhance        stability of the duplex. It is preferred that pairings which        increase the propensity to form a duplex are used at 1 or more        of the positions in the duplex at the 3′ end of the AS strand.        The terminal pair (the most 3′ pair in terms of the AS strand)        is designated as P₁, and the subsequent pairing positions (going        in the 5′ direction in terms of the AS strand) in the duplex are        designated, P₂, P₃, P₄, P₅, and so on. The preferred region in        which to modify to modulate duplex formation is at P₅ through        P₁, more preferably P₄ through P₁, more preferably P₃ through        P₁. Modification at P₁, is particularly preferred, alone or with        modification(s) at other position(s), e.g., any of the positions        just identified. It is preferred that at least 1, and more        preferably 2, 3, 4, or 5 of the pairs of the recited regions be        chosen independently from the group of:    -   G:C    -   a pair having an analog that increases stability over        Watson-Crick matches (A:T, A:U, G:C)    -   2-amino-A:U        2-thio U or 5 Me-thio-U:A    -   G-clamp (an analog of C having 4 hydrogen bonds):G    -   guanadinium-G-clamp:G    -   pseudo uridine:A    -   a pair in which one or both subunits has a sugar modification,        e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance        binding.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairs which promote duplex stability.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are G:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are a pair having an analog that increases stability overWatson-Crick matches.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are 2-amino-A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are 2-thio U or 5 Me-thio-U:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are guanidinium-G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are pseudo uridine:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are a pair in which one or both subunits has a sugarmodification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, whichenhances binding.

G-clamps and guanidinium G-clamps are discussed in the followingreferences: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-ClampContaining Oligonucleotides and Their Inhibition of the HIV-1 Tat-TARInteraction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262,2003; Holmes et al., “Steric inhibition of human immunodeficiency virustype-1 Tat-dependent trans-activation in vitro and in cells byoligonucleotides containing 2′-O-methyl G-clamp ribonucleosideanalogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al.,“Structural basis for recognition of guanosine by a synthetic tricycliccytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta,86:966-978, 2003; Rajeev, et al., “High-Affinity Peptide Nucleic AcidOligomers Containing Tricyclic Cytosine Analogues,” Organic Letters,4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- andGuanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002;Maier et al., “Nuclease resistance of oligonucleotides containing thetricyclic cytosine analogues phenoxazine and9-(2-aminoethoxy)-phenoxazine (“G-clamp”) and origins of their nucleaseresistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al.,“A cytosine analog that confers enhanced potency to antisenseoligonucleotides,” Proceedings Of The National Academy Of Sciences OfThe United States Of America, 96:3513-8, 1999.

Simultaneously Decreasing the Stability of the AS 5′ end of the Duplexand Increasing the Stability of the AS 3′ End of the Duplex

As is discussed above, an iRNA agent can be modified to both decreasethe stability of the AS 5′ end of the duplex and increase the stabilityof the AS 3′ end of the duplex. This can be effected by combining one ormore of the stability decreasing modifications in the AS 5′ end of theduplex with one or more of the stability increasing modifications in theAS 3′ end of the duplex. Accordingly a preferred embodiment includesmodification in P⁻⁵ through P⁻¹, more preferably P⁻⁴ through P⁻¹ andmore preferably P⁻³ through P⁻¹. Modification at P⁻¹, is particularlypreferred, alone or with other position, e.g., the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions of the AS 5′ end ofthe duplex region be chosen independently from the group of:

-   -   A:U    -   G:U    -   I:C    -   mismatched pairs, e.g., non-canonical or other than canonical        pairings which include a universal base; and

a modification in P₅ through P₁, more preferably P₄ through P₁ and morepreferably P₃ through P₁. Modification at P_(i), is particularlypreferred, alone or with other position, e.g., the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions of the AS 3′ end ofthe duplex region be chosen independently from the group of:

-   -   G:C    -   a pair having an analog that increases stability over        Watson-Crick matches (A:T, A:U, G:C)    -   2-amino-A:U        2-thio U or 5 Me-thio-U:A    -   G-clamp (an analog of C having 4 hydrogen bonds):G    -   guanadinium-G-clamp:G    -   pseudo uridine:A    -   a pair in which one or both subunits has a sugar modification,        e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance        binding.

The invention also includes methods of selecting and making iRNA agentshaving DMTDS. E.g., when screening a target sequence for candidatesequences for use as iRNA agents one can select sequences having a DMTDSproperty described herein or one which can be modified, preferably withas few changes as possible, especially to the

AS strand, to provide a desired level of DMTDS.

The invention also includes, providing a candidate iRNA agent sequence,and modifying at least one P in P⁻⁵ through P⁻¹ and/or at least one P inP₅ through P₁ to provide a DMTDS iRNA agent.

DMTDS iRNA agents can be used in any method described herein, e.g., tosilence an htt RNA, to treat any disorder described herein, e.g., aneurological disorder, in any formulation described herein, andgenerally in and/or with the methods and compositions describedelsewhere herein. DMTDS iRNA agents can incorporate other modificationsdescribed herein, e.g., the attachment of targeting agents or theinclusion of modifications which enhance stability, e.g., the inclusionof nuclease resistant monomers or the inclusion of single strandoverhangs (e.g., 3′ AS overhangs and/or 3′ S strand overhangs) whichself associate to form intrastrand duplex structure.

Preferably these iRNA agents will have an architecture described herein.

Other Embodiments

An RNA, e.g., an mRNA agent, can be produced in a cell in vivo, e.g.,from exogenous DNA templates that are delivered into the cell. Forexample, the DNA templates can be inserted into vectors and used as genetherapy vectors. Gene therapy vectors can be delivered to a subject by,for example, intravenous injection, local administration (U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al., Proc.Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. The DNA templates, for example, caninclude two transcription units, one that produces a transcript thatincludes the top strand of an iRNA agent and one that produces atranscript that includes the bottom strand of an iRNA agent. When thetemplates are transcribed, the iRNA agent is produced, and processedinto sRNA agent fragments that mediate gene silencing.

Physiological Effects

The iRNA agents described herein can be designed such that determiningtherapeutic toxicity is made easier by the complementarity of the iRNAagent with both a human and a non-human animal sequence. By thesemethods, an iRNA agent can consist of a sequence that is fullycomplementary to a nucleic acid sequence from a human and a nucleic acidsequence from at least one non-human animal, e.g., a non-human mammal,such as a rodent, ruminant or primate. For example, the non-human mammalcan be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus,Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence ofthe iRNA agent could be complementary to sequences within homologousgenes, e.g., oncogenes or tumor suppressor genes, of the non-humanmammal and the human. By determining the toxicity of the iRNA agent inthe non-human mammal, one can extrapolate the toxicity of the iRNA agentin a human. For a more strenuous toxicity test, the iRNA agent can becomplementary to a human and more than one, e.g., two or three or more,non-human animals.

The methods described herein can be used to correlate any physiologicaleffect of an iRNA agent on a human, e.g., any unwanted effect, such as atoxic effect, or any positive, or desired effect.

Amphipathic Delivery Agents

An mRNA agent, described herein can be used with an amphipathic deliveryconjugate or module, such as those described herein.

An amphipathic molecule is a molecule having a hydrophobic and ahydrophilic region. Such molecules can interact with (e.g., penetrate ordisrupt) lipids, e.g., a lipid bilayer of a cell. As such, they canserve as delivery agent for an associated (e.g., bound) iRNA (e.g., aniRNA or sRNA described herein). A preferred amphipathic molecule to beused in the compositions described herein (e.g., the amphipathic iRNAconstructs described herein) is a polymer. The polymer may have asecondary structure, e.g., a repeating secondary structure.

One example of an amphipathic polymer is an amphipathic polypeptide,e.g., a polypeptide having a secondary structure such that thepolypeptide has a hydrophilic and a hybrophobic face. The design ofamphipathic peptide structures (e.g., alpha-helical polypeptides) isroutine to one of skill in the art. For example, the followingreferences provide guidance: Grell et al. (2001) J Pept Sci 7(3):146-51;Chen et al. (2002) J Pept Res 59(1):18-33; Iwata et al. (1994) J BiolChem 269(7):4928-33; Cornut et al. (1994) FEBS Lett 349(1):29-33;Negrete et al. (1998) Protein Sci 7(6):1368-79.

Another example of an amphipathic polymer is a polymer made up of two ormore amphipathic subunits, e.g., two or more subunits containing cyclicmoieties (e.g., a cyclic moiety having one or more hydrophilic groupsand one or more hydrophobic groups). For example, the subunit maycontain a steroid, e.g., cholic acid; or a aromatic moiety. Suchmoieties preferably can exhibit atropisomerism, such that they can formopposing hydrophobic and hydrophilic faces when in a polymer structure.

The ability of a putative amphipathic molecule to interact with a lipidmembrane, e.g., a cell membrane, can be tested by routine methods, e.g.,in a cell free or cellular assay. For example, a test compound iscombined or contacted with a synthetic lipid bilayer, a cellularmembrane fraction, or a cell, and the test compound is evaluated for itsability to interact with, penetrate, or disrupt the lipid bilayer, cellmembrane or cell. The test compound can be labeled in order to detectthe interaction with the lipid bilayer, cell membrane, or cell. Inanother type of assay, the test compound is linked to a reportermolecule or an iRNA agent (e.g., an iRNA or sRNA described herein), andthe ability of the reporter molecule or iRNA agent to penetrate thelipid bilayer, cell membrane or cell is evaluated. A two-step assay canalso be performed, wherein a first assay evaluates the ability of a testcompound alone to interact with a lipid bilayer, cell membrane or cell;and a second assay evaluates the ability of a construct (e.g., aconstruct described herein) that includes the test compound and areporter or iRNA agent to interact with a lipid bilayer, cell membraneor cell.

An amphipathic polymer useful in the compositions described herein hasat least 2, preferably at least 5, more preferably at least 10, 25, 50,100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids orcyclic subunits). A single amphipathic polymer can be linked to one ormore, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agentsdescribed herein). In some embodiments, an amphipathic polymer cancontain both amino acid and cyclic subunits, e.g., aromatic subunits.

The invention features a composition that includes an iRNA agent (e.g.,an iRNA or sRNA described herein) in association with an amphipathicmolecule. Such compositions may be referred to herein as “amphipathiciRNA constructs.” Such compositions and constructs are useful in thedelivery or targeting of iRNA agents, e.g., delivery or targeting ofiRNA agents to a cell. While not wanting to be bound by theory, suchcompositions and constructs can increase the porosity of, e.g., canpenetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g.,to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNAagent (e.g., an iRNA or sRNA agent described herein) linked to anamphipathic molecule. The iRNA agent and the amphipathic molecule may beheld in continuous contact with one another by either covalent ornoncovalent linkages.

The amphipathic molecule of the composition or construct is preferablyother than a phospholipid, e.g., other than a micelle, membrane ormembrane fragment.

The amphipathic molecule of the composition or construct is preferably apolymer. The polymer may include two or more amphipathic subunits. Oneor more hydrophilic groups and one or more hydrophobic groups may bepresent on the polymer. The polymer may have a repeating secondarystructure as well as a first face and a second face. The distribution ofthe hydrophilic groups and the hydrophobic groups along the repeatingsecondary structure can be such that one face of the polymer is ahydrophilic face and the other face of the polymer is a hydrophobicface.

The amphipathic molecule can be a polypeptide, e.g., a polypeptidecomprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunitscontaining one or more cyclic moiety (e.g., a cyclic moiety having oneor more hydrophilic groups and/or one or more hydrophobic groups). Inone embodiment, the polymer is a polymer of cyclic moieties such thatthe moieties have alternating hydrophobic and hydrophilic groups. Forexample, the subunit may contain a steroid, e.g., cholic acid. Inanother example, the subunit may contain an aromatic moiety. Thearomatic moiety may be one that can exhibit atropisomerism, e.g., a2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted)biphenyl. A subunit may include an aromatic moiety of Formula (M):

The invention features a composition that includes an iRNA agent (e.g.,an iRNA or sRNA described herein) in association with an amphipathicmolecule. Such compositions may be referred to herein as “amphipathiciRNA constructs.” Such compositions and constructs are useful in thedelivery or targeting of iRNA agents, e.g., delivery or targeting ofiRNA agents to a cell. While not wanting to be bound by theory, suchcompositions and constructs can increase the porosity of, e.g., canpenetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g.,to allow entry of the iRNA agent into a cell.

Referring to Formula M, R₁ is C₁-C₁₀₀ alkyl optionally substituted witharyl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted withO, S, alkenyl or alkynyl; C₁-C₁₀₀ perfluoroalkyl; or OR₅.

R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonicacid; OR₆; or C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo,nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonicacid, phosphate, phosphate ester, substituted or unsubstituted aryl,carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/oroptionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

R₃ is hydrogen, or when taken together with R₄ forms a fused phenylring.

R₄ is hydrogen, or when taken together with R₃ forms a fused phenylring.

R₅ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl,alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl;or C₁-C₁₀₀ perfluoroalkyl; and R₆ is C₁-C₁₀₀ alkyl optionallysubstituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl oralkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester,substituted or unsubstituted aryl, carboxyl, carboxylate, aminocarbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S,S(O), SO₂, alkenyl, or alkynyl.

An iRNA agent can have a ZXY structure, such as is described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

The sense and antisense sequences of an iRNA agent can be palindromic.Exemplary features of palindromic iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a deliveryagent that features a modular complex. The complex can include a carrieragent linked to one or more of (preferably two or more, more preferablyall three of): (a) a condensing agent (e.g., an agent capable ofattracting, e.g., binding, a nucleic acid, e.g., through ionic orelectrostatic interactions); (b) a fusogenic agent (e.g., an agentcapable of fusing and/or being transported through a cell membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type. iRNA agents complexed to a delivery agent aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An mRNA agent can have non-canonical pairings, such as between the senseand antisense sequences of the iRNA duplex. Exemplary features ofnon-canonical iRNA agents are described in co-owned PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004.

Increasing Cellular Uptake of dsRNAs

A method of the invention that can include the administration of an iRNAagent and a drug that affects the uptake of the iRNA agent into thecell. The drug can be administered before, after, or at the same timethat the iRNA agent is administered. The drug can be covalently linkedto the iRNA agent. The drug can have a transient effect on the cell.

The drug can increase the uptake of the iRNA agent into the cell, forexample, by disrupting the cell's cytoskeleton, e.g., by disrupting thecell's microtubules, microfilaments, and/or intermediate filaments. Thedrug can be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

iRNA Conjugates

An iRNA agent conjugated to lipophilic agent for enhanced uptake into aneural cell can be coupled, e.g., covalently coupled, to a second agent.For example, an iRNA agent used to treat a particular neurologicaldisorder can be coupled to a second therapeutic agent, e.g., an agentother than the iRNA agent. The second therapeutic agent can be one whichis directed to the treatment of the same neurological disorder. Forexample, in the case of an iRNA used to treat a HD, the iRNA agent canbe coupled to a second agent which is known to be useful for thetreatment of HD.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods.Exemplary methods include: organic synthesis and RNA cleavage, e.g., invitro cleavage.

Organic Synthesis. An iRNA can be made by separately synthesizing eachrespective strand of a double-stranded RNA molecule. The componentstrands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB(Uppsala Sweden), can be used to produce a large amount of a particularRNA strand for a given iRNA. The OligoPilotII reactor can efficientlycouple a nucleotide using only a 1.5 molar excess of a phosphoramiditenucleotide. To make an RNA strand, ribonucleotides amidites are used.Standard cycles of monomer addition can be used to synthesize the 21 to23 nucleotide strand for the iRNA. Typically, the two complementarystrands are produced separately and then annealed, e.g., after releasefrom the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. Thecomplementary of the species to a particular target gene can beprecisely specified. For example, the species may be complementary to aregion that includes a polymorphism, e.g., a single nucleotidepolymorphism. Further the location of the polymorphism can be preciselydefined. In some embodiments, the polymorphism is located in an internalregion, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of thetermini.

dsRNA Cleavage. iRNAs can also be made by cleaving a larger ds iRNA. Thecleavage can be mediated in vitro or in vivo. For example, to produceiRNAs by cleavage in vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid(DNA) segment in both directions. For example, the HiScribe™ RNAitranscription kit (New England Biolabs) provides a vector and a methodfor producing a dsRNA for a nucleic acid segment that is cloned into thevector at a position flanked on either side by a T7 promoter. Separatetemplates are generated for T7 transcription of the two complementarystrands for the dsRNA. The templates are transcribed in vitro byaddition of T7 RNA polymerase and dsRNA is produced. Similar methodsusing PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) canalso be used. In one embodiment, RNA generated by this method iscarefully purified to remove endotoxins that may contaminatepreparations of the recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example,using a Dicer or comparable RNAse III-based activity. For example, thedsRNA can be incubated in an in vitro extract from Drosophila or usingpurified components, e.g. a purified RNAse or RISC complex (RNA-inducedsilencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15;15(20):2654-9. and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, eachbeing a particular 21 to 23 nt fragment of a source dsRNA molecule. Forexample, iRNAs that include sequences complementary to overlappingregions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Synthesis of modified and nucleotide surrogate iRNA agents is discussedbelow.

Formulation

The iRNA agents described herein can be formulated for administration toa subject.

For ease of exposition, the formulations, compositions, and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions, and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration.

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A mRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA that can mediated RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least three, five, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than an RNA or a DNA). Forexample, a iRNA composition for the treatment of a neurological disease,e.g., neurodegenerative disease, such as PD, might include a known PDtherapeutic (e.g., levadopa or depronil)

Targeting

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAs. Itshould be understood, however, that these formulations, compositions andmethods can be practiced with other iRNA agents, e.g., modified iRNAagents, and such practice is within the invention.

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent,or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which canbe processed into a sRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)is targeted to a particular cell. For example, a liposome or particle orother structure that includes a iRNA can also include a targeting moietythat recognizes a specific molecule on a target cell. The targetingmoiety can be a molecule with a specific affinity for a target cell.Targeting moieties can include antibodies directed against a proteinfound on the surface of a target cell, or the ligand or areceptor-binding portion of a ligand for a molecule found on the surfaceof a target cell.

An antigen, can be used to target an iRNA to a neural cell in the brain.

In one embodiment, the targeting moiety is attached to a liposome. Forexample, U.S. Pat. No. 6,245,427 describes a method for targeting aliposome using a protein or peptide. In another example, a cationiclipid component of the liposome is derivatized with a targeting moiety.For example, WO 96/37194 describes convertingN-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimideactivated ester. The product was then coupled to an RGD peptide.

Treatment Methods and Routes of Delivery

A composition that includes an iRNA agent targeting a gene expressed inneural cells, can be delivered to a subject by a variety of routes.Exemplary routes include intrathecal, parenchymal (e.g., in the brain),nasal, and ocular delivery. The composition can also be deliveredsystemically, e.g., by intravenous, subcutaneous or intramuscularinjection, which is particularly useful for delivery of the iRNA agentsto peripheral neurons. A preferred route of delivery is directly to thebrain, e.g., into the ventricles or the hypothalamus of the brain, orinto the lateral or dorsal areas of the brain. The iRNA agents forneural cell delivery can be incorporated into pharmaceuticalcompositions suitable for administration. For example, compositions caninclude one or more species of an iRNA agent and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. A pharmaceutically acceptable carrierdoes not include a transfection reagent or a reagent to facilitateuptake in a neural cell that is in addition to the lipophilic moietyconjugated to the iRNA agent featured in the invention. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, intranasal,transdermal), oral or parenteral. Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, intrathecal, or intraventricular (e.g.,intracerebroventricular) administration.

The route of delivery can be dependent on the disorder of the patient.For example, a subject diagnosed with HD can be administered an anti-httiRNA agent conjugated to a lipophilic agent directly into the brain(e.g., into the globus pallidus or the corpus striatum of the basalganglia, and near the medium spiny neurons of the corpos striatum). Asubject diagnosed with multiple system atrophy can be administered aniRNA agent directly into the brain, e.g., into the striatum andsubstantia nigra regions of the brain, and into the spinal cord. Asubject diagnosed with Lewy body dementia can be administered an iRNAagent directly into the brain, e.g., directly into the cortex of thebrain, and administration can be diffuse. In addition to an iRNA agentmodified for enhanced delivery to neural cells, a patient can beadministered a second therapy, e.g., a palliative therapy and/ordisease-specific therapy. The secondary therapy can be, for example,symptomatic, (e.g., for alleviating symptoms), neuroprotective (e.g.,for slowing or halting disease progression), or restorative (e.g., forreversing the disease process). Preferable, the subject is notadministered an anti-SNCA iRNA.

For the treatment of HD, for example, symptomatic therapies can includethe drugs haloperidol, carbamazepine, or valproate. Other therapies caninclude psychotherapy, physiotherapy, speech therapy, communicative andmemory aids, social support services, and dietary advice.

For the treatment of Parkinson's Disease, symptomatic therapies caninclude the drugs carbidopa/levodopa, entacapone, tolcapone,pramipexole, ropinerole, pergolide, bromocriptine, selegeline,amantadine, and several anticholingergic agents. Deep brain stimulationsurgery as well as stereotactic brain lesioning may also providesymptomatic relief. Neuroprotective therapies include, for example,carbidopa/levodopa, selegeline, vitamin E, amantadine, pramipexole,ropinerole, coenzyme Q10, and GDNF. Restorative therapies can include,for example, surgical transplantation of stem cells.

An iRNA agent conjugated with a lipophilic moiety can be delivered toneural cells of the brain. Delivery methods that do not require passageof the composition across the blood-brain barrier can be utilized. Forexample, a pharmaceutical composition containing an iRNA agent can bedelivered to the patient by injection directly into the area containingthe disease-affected cells. For example, the pharmaceutical compositioncan be delivered by injection directly into the brain. The injection canbe by stereotactic injection into a particular region of the brain(e.g., the substantia nigra, cortex, hippocampus, striatum, or globuspallidus). The iRNA agent can be delivered into multiple regions of thecentral nervous system (e.g., into multiple regions of the brain, and/orinto the spinal cord). The iRNA agent can be delivered into diffuseregions of the brain (e.g., diffuse delivery to the cortex of thebrain).

In one embodiment, the iRNA agent can be delivered by way of a cannulaor other delivery device having one end implanted in a tissue, e.g., thebrain, e.g., the substantia nigra, cortex, hippocampus, striatum orglobus pallidus of the brain. The cannula can be connected to areservoir of iRNA agent. The flow or delivery can be mediated by a pump,e.g., an osmotic pump or minipump, such as an Alzet pump (Durect,Cupertino, Calif.). In one embodiment, a pump and reservoir areimplanted in an area distant from the tissue, e.g., in the abdomen, anddelivery is effected by a conduit leading from the pump or reservoir tothe site of release. Devices for delivery to the brain are described,for example, in U.S. Pat. Nos. 6,093,180, and 5,814,014.

An iRNA agent conjugated to a lipophilic moiety, e.g., cholesterol, canbe further modified such that it is capable of traversing the bloodbrain barrier. For example, the iRNA agent can be conjugated to amolecule that enables the agent to traverse the barrier. Such modifiediRNA agents can be administered by any desired method, such as byintraventricular or intramuscular injection, or by pulmonary delivery,for example.

The iRNA agent conjugated to a lipophilic moiety for enhanced uptakeinto neural cells can be administered ocularly, such as to treat retinaldisorder, e.g., a retinopathy. For example, the pharmaceuticalcompositions can be applied to the surface of the eye or nearby tissue,e.g., the inside of the eyelid. They can be applied topically, e.g., byspraying, in drops, as an eyewash, or an ointment. Ointments ordroppable liquids may be delivered by ocular delivery systems known inthe art such as applicators or eye droppers. Such compositions caninclude mucomimetics such as hyaluronic acid, chondroitin sulfate,hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives suchas sorbic acid, EDTA or benzylchronium chloride, and the usualquantities of diluents and/or carriers. The pharmaceutical compositioncan also be administered to the interior of the eye, and can beintroduced by a needle or other delivery device which can introduce itto a selected area or structure. The composition containing the iRNAagent can also be applied via an ocular patch.

Administration can be provided by the subject or by another person,e.g., a another caregiver. A caregiver can be any entity involved withproviding care to the human: for example, a hospital, hospice, doctor'soffice, outpatient clinic; a healthcare worker such as a doctor, nurse,or other practitioner; or a spouse or guardian, such as a parent. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose.

The subject can be monitored for reactions to the treatment, such asedema or hemorrhaging. For example, the patient can be monitored by MRI,such as daily or weekly following injection, and at periodic timeintervals following injection.

The subject can also be monitored for an improvement or stabilization ofdisease symptoms. Such monitoring can be achieved, for example, byserial clinical assessments (e.g., using the United Parkinson's DiseaseRating Scale) or functional neuroimaging. Monitoring can also includeserial quantitative measures of striatal dopaminergic function (e.g.,fluorodopa and positron emission tomography) comparing treated subjectsto normative data collected from untreated subjects. Additional outcomemeasures can include survival and survival free of palliative therapyand nursing home placement. Statistically significant differences inthese measurements and outcomes for treated and untreated subjects isevidence of the efficacy of the treatment.

A pharmaceutical composition containing an iRNA agent conjugated to alipophilic moiety for enhanced uptake into neural cells can beadministered to any patient diagnosed as having or at risk fordeveloping a neurological disorder, such as HD. In one embodiment, thepatient is diagnosed as having a neurological disorder, and the patientis otherwise in general good health. For example, the patient is notterminally ill, and the patient is likely to live at least 2, 3, 5, or10 years or longer following diagnosis. The patient can be treatedimmediately following diagnosis, or treatment can be delayed until thepatient is experiencing more debilitating symptoms, such as motorfluctuations and dyskinesis in PD patients. In another embodiment, thepatient has not reached an advanced stage of the disease, e.g., thepatient has not reached Hoehn and Yahr stage 5 of PD (Hoehn and Yahr,Neurology 17:427-442, 1967). In general, an iRNA agent conjugated to alipophilic moiety can be administered by any suitable method. As usedherein, topical delivery can refer to the direct application of an iRNAagent to any surface of the body, including the eye, a mucous membrane,surfaces of a body cavity, or to any internal surface. Formulations fortopical administration may include transdermal patches, ointments,lotions, creams, gels, drops, sprays, and liquids. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Topical administration can alsobe used as a means to selectively deliver the iRNA agent to theepidermis or dermis of a subject, or to specific strata thereof, or toan underlying tissue.

Compositions for intrathecal or intraventricular (e.g.,intracerebroventricular) administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Compositions for intrathecal or intraventricularadministration preferably do not include a transfection reagent or anadditional lipophilic moiety besides the lipophilic moiety attached tothe iRNA agent.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

An iRNA agent conjugated to a lipophilic agent for enhanced uptake intoneural cells can be administered to a subject by pulmonary delivery.Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably iRNA, withinthe dispersion can reach the lung where it can be readily absorbedthrough the alveolar region directly into blood circulation. Pulmonarydelivery can be effective both for systemic delivery and for localizeddelivery to treat diseases of the lungs. In one embodiment, an iRNAagent administered by pulmonary delivery has been modified such that itis capable of traversing the blood brain barrier.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An iRNAcomposition may be stably stored as lyophilized or spray-dried powdersby itself or in combination with suitable powder carriers. The deliveryof a composition for inhalation can be mediated by a dosing timingelement which can include a timer, a dose counter, time measuringdevice, or a time indicator which when incorporated into the deviceenables dose tracking, compliance monitoring, and/or dose triggering toa patient during administration of the aerosol medicament.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

An iRNA agent conjugated to a lipophilic moiety for enhanced uptake intoneural cells can be administered by oral and nasal delivery. Forexample, drugs administered through these membranes have a rapid onsetof action, provide therapeutic plasma levels, avoid first pass effect ofhepatic metabolism, and avoid exposure of the drug to the hostilegastrointestinal (GI) environment. Additional advantages include easyaccess to the membrane sites so that the drug can be applied, localizedand removed easily. In one embodiment, an iRNA agent administered byoral or nasal delivery has been modified to be capable of traversing theblood-brain barrier.

In one embodiment, unit doses or measured doses of a composition thatinclude iRNA are dispensed by an implanted device. The device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include a pump, such as an osmotic pump and,optionally, associated electronics.

An iRNA agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

Dosage. An iRNA agent modified for enhance uptake into neural cells canbe administered at a unit dose less than about 1.4 mg per kg ofbodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, andless than 200 nmole of RNA agent (e.g., about 4.4×10¹⁶ copies) per kg ofbodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75,0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agentper kg of bodyweight. The unit dose, for example, can be administered byinjection (e.g., intravenous or intramuscular, intrathecally, ordirectly into the brain), an inhaled dose, or a topical application.Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of bodyweight.

Delivery of an iRNA agent directly to an organ (e.g., directly to thebrain) can be at a dosage on the order of about 0.00001 mg to about 3 mgper organ, or preferably about 0.0001-0.001 mg per organ, about 0.03-3.0mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.

The dosage can be an amount effective to treat or prevent a neurologicaldisease or disorder, e.g., HD.

In one embodiment, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject hasPD and the modality is a therapeutic agent other than an iRNA agent,e.g., other than a double-stranded iRNA agent, or sRNA agent. Thetherapeutic modality can be, for example, levadopa or depronil.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into an sRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, orprecursor thereof). The maintenance dose or doses are generally lowerthan the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g.,10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. Themaintenance doses are preferably administered no more than once every 5,10, or 30 days. Further, the treatment regimen may last for a period oftime which will vary depending upon the nature of the particulardisease, its severity and the overall condition of the patient. Inpreferred embodiments the dosage may be delivered no more than once perday, e.g., no more than once per 24, 36, 48, or more hours, e.g., nomore than once every 5 or 8 days. Following treatment, the patient canbe monitored for changes in his condition and for alleviation of thesymptoms of the disease state. The dosage of the compound may either beincreased in the event the patient does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disease state is observed, if the disease state hasbeen ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. In another embodiment, the iRNA agentspecies has sequences that are non-overlapping and non-adjacent toanother species with respect to a naturally occurring target sequence.In another embodiment, the plurality of iRNA agent species is specificfor different naturally occurring target genes. In another embodiment,the iRNA agent is allele specific.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. nasal, buccal, orpulmonary. For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) can include a single treatment or,preferably, can include a series of treatments. It will also beappreciated that the effective dosage of an iRNA agent such as an sRNAagent used for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentfrom the results of diagnostic assays as described herein. For example,the subject can be monitored after administering an iRNA agentcomposition. Based on information from the monitoring, an additionalamount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target RNA, e.g., an RNA expressed in a neural cell. Thetransgenic animal can be deficient for the corresponding endogenous RNA.In another embodiment, the composition for testing includes an iRNAagent that is complementary, at least in an internal region, to asequence that is conserved between the target RNA in the animal modeland the target RNA in a human.

Kits. In certain other aspects, the invention provides kits that includea suitable container containing a pharmaceutical formulation of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). In certain embodimentsthe individual components of the pharmaceutical formulation may beprovided in one container. Alternatively, it may be desirable to providethe components of the pharmaceutical formulation separately in two ormore containers, e.g., one container for an iRNA agent preparation, andat least another for a carrier compound. The kit may be packaged in anumber of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 Cholesterol Conjugated siRNAs are taken up intoPrimary Striatal Neurons

Primary striatal neurons were isolated from mouse fetal tissue at day15.5 gestation. The isolated cells were cultured in NeuroBasal™ medium(Gibco) for 7 days. An siRNA conjugated with cholesterol and Cy3 (calledChol-siRNA-Cy3) targeting against GFP, in a solution of PBS, wasintroduced to a culture of primary striatal neurons isolated from mouse(final concentration=50 nM). The cells were incubated withchol-siRNA-Cy3 for 6-12 hours and then the medium was changed, washingaway any chol-siRNA-Cy3 that was not taken up into the cell. Notransfection agents were used. Nearly all primary neurons were observedto contain the chol-siRNA-Cy3 in the cytoplasm of neurons. An siRNA-Cy3(without a cholesterol conjugate) was found to be taken up by primaryneurons to a much lesser extent then the cholesterol-conjugated siRNAwhen the cells were cultured under the same conditions.

Example 2 Cholesterol-conjugated siRNAs were Administered to Neurons InVivo

We administered chol-siRNA-Cy3 against GFP in mouse striatum by a singledirect injection and by Alzet® pump (DURECT Corporation, Cupertino,Calif.) over 7 days. The direct injection contained 50 μM in 1 μlsolution with PBS, and the Alzet pump delivered 50 μM in 1 μl per day.We compared the chol-siRNA with high dose unconjugated siRNA-Cy3, at thesame doses. By fluorescence microscopy, we found that both sets ofsiRNAs entered many brain cells. The chol-siRNA-Cy3 had a higherfrequency of cell entry than the unconjugated siRNA in administrationwith Alzet pump by observation. Direct injection of chol-siRNA-Cy3showed presence of Cy3 siRNA one week later, whereas direct injection ofunconjugated siRNA showed very little Cy3 labeling after one week.

In separate experiments, 2 μl of 50 μM chol-siRNA-Cy3 in PBS wasinjected into the striatum of mice. Three days later, mice were perfusedand striatal sections prepared for immunofluorescence (FITC) for DARRP32. DARRP 32 serves as a marker for medium spiny neurons, a neuronaltype affected in Huntington's disease. The data indicated that thechol-siRNA at the volume tested spreads throughout the extent of thestriatum and enters medium size spiny neurons. The sections were studiedunder 60× oil, to ensure that the Cy3 labeling resides inside theneurons, not on the surface. Sections at each of the three striatalregions were counted (1113 cells in all). 98% of the cells in eachstriatal region had colocalization of FITC (DARRP 32) and Cy3(chol-siRNA). These pilot studies provide support that modified siRNAcan be delivered to brain, to enter neurons.

To investigate whether cholesterol conjugated siRNAs were toxic to thestriatal cells in vivo, the cells were stained with fluorojade, a markerfor apoptotic cell death. Fluorojade staining was observed along theinjection site, but not in the surrounding cells. In a positive controlexperiment, fluorojade staining was observed in striatal cells in vivofollowing injection of the NMDA receptor agonist quinolinic acid, whichis known to induce neuronal cell death. These experiments indicated thatchol-siRNA is not toxic to striatal cells in vivo.

Example 3 GFP Expression was Inhibited in PC12 Cells Stably Transfectedwith GFP-htt

We tested whether chol-siRNA targeting GFP could knockdown GFPexpression, and also examined the duration of this RNAi activity. Weadded chol-siRNA versus GFP (50 nM final concentration) to PC12 cellsstably transfected with GFP fused to human mut-htt carrying about a 100Q expansion (Qin et al., Hum Mol. Genet. 12:3231-44, 2003, Epub 2003Oct. 21). Treatment with pronasterone increases expression of a becauseof promoter GFP-htt protein, but significantly reduces GFP fluorescencein the stably transfected cells. A control pronasterone-treated PC12culture was treated with chol-siRNA in PBS (final concentration=100 nM)targeting luciferase; and a test pronasterone-treated PC12 cell culturewas treated with chol-siRNA (final concentration 100 nM) targeting GFP.No transfection reagents were used. The chol-siRNA was kept in theculture medium for 6-12 hours and then the medium was refreshed,therefore washing out an chol-siRNA not incorporated into the cells.Images were taken one week later to assess the effect on cellfluorescence. GFP fluorescence was decreased to a much greater extent incells treated with chol-siRNA targeting GFP, than in cells treated withchol-siRNA targeting luciferase.

Example 4 siRNA Protects against Huntingtin-induced Neuronal Dysfunction

With evidence that chol-siRNA can enter brain cells and knockdown targetgene expression (see above), we tested chol-siRNA against human mut-httcarrying about a 100-Q expansion expression in vivo. In a mouse model ofHuntington's Disease, introduction of lentivirus-mut-htt (1 μl, 1×10¹⁰particles) leads to clasping 5 days later. We injectedlentivirus-mut-htt into the cortex and striatum of 4 mice. In two of themice, we co-injected chol-siRNA against htt mRNA. In one mouse, weco-injected chol-siRNA targeting luciferase, and in the other mouse, weco-injected vehicle. The two mice that received chol-siRNA against httshowed no clasping at 7 days. The control mice clasped, as expected. Theresults are shown in Table 8.

TABLE 8 Study Testing Cholesterol siRNA in Vivo Animal Treatment 1 2 3 4Lentivirus-mut-htt + + + + Chol-siRNA targeting mut-htt + + − −Chol-siRNA targeting GFP − − + − No siRNA − − − + Behavior: Clasping NoNo Yes Yes

Example 5 Hallmarks of Huntington's Disease are Found in Mice Treatedwith Lentivirus-mut-htt

Mice continue to have clasping for seven months after lentivirus-mut-httadministration into a unilateral striatum. In other respects, the micegrew and moved as expected. Furthermore, mice treated withlentivirus-WT-htt (CAG repeat of 18) show no evidence of clasping overthe course of the experimental protocol, which was to three weekspost-injection. The images in FIGS. 1A-1D are taken from the striatum ofa mouse seven months after injection with lentivirus-mut-htt. The tissuewas treated with an antiserum against the N-terminus of huntingtin (Abl)for immunohistochemical analysis. Notable phenotypes include nuclearinclusions (arrowheads) and dystrophic neurites (arrows) similar tothose found in adult-onset human HD (see DiFiglia et al, Science277:1990-3, 1997). Use of the lentivirus model is convenient for usewith co-injection of siRNA. Coinjection allows for reduced discrepanciesin time and space that may complicate delivery of siRNA in transgenicmouse models.

Example 6 A single Intrastriatal Administration of siRNA TargetingHuntingtin Reduces Neuropathology in a Mouse Model of Huntington'sDisease

The effect of an siRNA targeting huntingtin was evaluated in an AAVmouse model of Huntington's disease. In this mouse model of Huntington'sdisease, a portion of the mutant human huntingtin gene with apolyglutamine expansion comprising 100 CAG repeats is introduced intothe brain by viral (AAV) delivery. When a single intrastriatal injectionof 0.5 nmoles (7.5 ug) siRNA was administered, thecholesterol-conjugated siRNA, AL-DP-1799 (E1-4), targeting huntingtinreduced inclusion size in striatum (FIGS. 2, 3) and cortex (FIG. 3A),and reduced neuropil aggregates in striatum (FIG. 3B), compared with anon-physiological siRNA, AL-DP-1956, targeting luciferase. In addition,the number of huntingtin-immunoreactive cells in the striatum wassignificantly increased, consistent with an increase in survival ofstriatal neurons after a single intrastriatal injection of AL-DP-1799(FIG. 3).

Adult female SJL/B6 mice, 6 months of age, received an injection of 3 uLof 1.1×10¹³ titer units of AAV-htt-100Q, together with 0.5 nmoles (7.5ug) siRNA. AAV-htt-100Q comprised AAV serotype 8, for delivery of theportion of the human huntingtin gene encoding amino acids 1-400, with a100 CAG repeat (100Q). The siRNA tested was either acholesterol-conjugated siRNA targeting huntingtin (AL-DP-1799, below) oran irrelevant cholesterol-conjugated siRNA targeting luciferase(AL-DP-1956). For each mouse, 0.5 uL of 1 mM siRNA was injectedunilaterally into the striatum at a rate of 100 mL/minute. The injectioncoordinates were AP+1.0 mm, Lateral+1.8 mm, Ventral 2.3 mm. Forimmunohistochemical analysis, mice were sacrificed 14 days after siRNAinjection, and perfused intracardially with 4% paraformaldehyde. Brainswere removed and vibratome frozen sections of 30 or 40 μm thickness werecut. The primary antibody against huntingtin, Ab1, that recognizes bothhuman and mouse huntingtin, was made as described previously (DiFigliaM, Sapp E, Chase K, Schwarz C, Meloni A, Young C, Martin E, VonsattelJ-P, Carraway R, Reeves S A, Boyce F M, Carraway R, and Aronin N:Huntingtin is a cytoplasmic protein associated with vesicles in humanand rat brain neurons. Neuron 14:1075-1081, 1995; Aronin N, Chase K,Young C, Sapp E, Schwarcz C, Matta N, Kornreich R, Sheth A,Landwehrmeyer B, Bird E, Vonsattel J-P, Smith T, Carraway R, Boyce F M,Beal M F, Young A B, Penney J B, and DiFiglia M: CAG expansion affectsthe expression of mutant huntingtin in the Huntington's disease brain.Neuron 15:1193-1201, 1995). Immunoabsorbed antiserum Abl was used at aconcentration of 1 μg/ml. The secondary antibody was a goat anti-rabbitantibody (Vector Laboratories, California) and used at 1:10,000. For DABhistological processing, a kit was used (Pierce Laboratory, Illinois).

Sequence of Cholesterol-Conjugated dsRNA AL-DP-1799 AL-DP- Numbersense: 5′-3′ antisense: 5′-3′ AL-DP- CsCCUGGAAAAGCUGAUGACUsUCAUCAGCUUUUCCA 1799 GsGsChol GGGsUsC Note: ′s′ represents aphosphorothioate bound inbetween neighboring bases, ′Chol′ representscholesterol-conjugate

Mice that received AAV-htt-100Q exhibited huntingtin-immunoreactivity inthe ipsilateral striatum and cortex, whether they receivedcholesterol-conjugated siRNA targeting luciferase (AL-DP-1956; FIG. 2A)or cholesterol-conjugated siRNA targeting huntingtin (AL-DP-1799; FIG.2D). However, the appearance of intracellular staining for huntingtin inmice that received AAV-htt-100Q was clearly different, in that the sizeof the inclusions appeared smaller in the ipsilateral striatum of micetreated with AL-DP-1799 (cholesterol-conjugated siRNA targetinghuntingtin, FIG. 2F) than in the ipsilateral striatum of mice treatedwith AL-DP-1956 (cholesterol-conjugated siRNA targeting luciferase, FIG.2C). The contralateral striatum exhibited faint staining for huntingtinin mice that received AAV-htt-100Q, whether they receivedcholesterol-conjugated siRNA targeting luciferase (AL-DP-1956; FIG. 2B)or cholesterol-conjugated siRNA targeting huntingtin (AL-DP-1799; FIG.2E). When ipsilateral striatal and cortical inclusion sizes werequantified (70-100 inclusions measured per mouse) in mice treated withAL-DP-1956 (n=8) and mice treated with AL-DP-1799 (n=8), inclusion sizewas significantly (p<0.02) reduced in mice treated with AL-DP-1799compared to mice treated with AL-DP-1956. Median inclusion sizes foripsilateral cortex and striatum are shown as scatter plots in FIG. 3A.Therefore, a single intrastriatal injection of cholesterol-conjugatedsiRNA targeting huntingtin results in reduced striatal and corticalpathology, and represents a novel approach to providing effectivetreatment of Huntington's disease.

Moreover, when the same mice were evaluated for neuropil aggregates inthe striatum (FIG. 3B), mice that received AAV-htt-100Q and AL-DP-1799(cholesterol-conjugated siRNA targeting huntingtin) exhibited anapproximately two-thirds reduction (p<0.02) in the number of neuropilaggregates compared with mice that received AAV-htt-100Q and AL-DP-1956(cholesterol-conjugated siRNA targeting luciferase). Total neuropilaggregates were counted in a 2500 um² area, using 6 sections per mouse.These data provide additional evidence that a single intrastriatalinjection of cholesterol-conjugated siRNA targeting huntingtin resultsin reduced neuropathology, and represents a novel approach to providingeffective treatment of Huntington's disease.

The number of huntingtin-immunoreactive cells was scored in cortex andstriatum of 8 mice that received AAV-htt-100Q and AL-DP-1799(cholesterol-conjugated siRNA targeting huntingtin, ‘Htt’) and 8 micethat received AAV-htt-100Q and AL-DP-1956 (cholesterol-conjugated siRNAtargeting luciferase, ‘luc’). In the striatum (FIG. 4), a statisticallysignificant increase (p<0.001) was found in the mean number of totalhuntingtin-immunoreactive cells per 2500 um² area in mice treated withAL-DP-1799 (cholesterol-conjugated siRNA targeting huntingtin) comparedto mice treated with AL-DP-1956 (cholesterol-conjugated siRNA targetingluciferase). In the cortex (FIG. 4), there was a trend towards anincreased mean number of total huntingtin-immunoreactive cells per 2500um² area in mice treated with AL-DP-1799 (cholesterol-conjugated siRNAtargeting huntingtin) compared to mice treated with AL-DP-1956(cholesterol-conjugated siRNA targeting luciferase). When cells withnuclear inclusions and cytoplasmic aggregates (‘+inc/+cyto’) were scoredseparately from cells with nuclear inclusions and no cytoplasmicaggregates (‘+inc/−cyto’), there was a statistically significantincrease (p<0.001) in the number of cells with nuclear inclusions andcytoplasmic aggregates in striatum. One explanation for this result isthat mice that received AAV-htt-100Q and were treated with AL-DP-1799(cholesterol-conjugated siRNA targeting huntingtin) have more survivingstriatal neurons. These data imply that a single intrastriatal injectionof cholesterol-conjugated siRNA targeting huntingtin results inprotection of striatal neurons, and represents a novel approach toproviding effective treatment of Huntington's disease.

Example 7 A Single Intrastriatal Administration of siRNA TargetingHuntingtin Reduces Abnormal Clasping Behavior in a Mouse Model ofHuntington's Disease

The effect of the siRNA targeting huntingtin was further evaluated inthe AAV mouse model of Huntington's disease by assessing claspingbehavior, a stereotypical and abnormal behavior characteristic of animalmodels of Huntington's disease. In the same mice where pathology wassubsequently evaluated, clasping was scored as a binary yes/no dailyassessment over a period of 14 days, and then the percentage of daysthat clasping was observed was determined for each mouse. The averagepercentage of clasping days was reduced by approximately half (p<0.01)in mice treated with the cholesterol-conjugated siRNA targetinghuntingtin, AL-DP-1799 ('HU', FIG. 4), as compared to mice that receiveda non-physiological siRNA targeting luciferase, AL-DP-1956 (‘Luc’, FIG.5). These data demonstrate that a single intrastriatal injection ofcholesterol-conjugated siRNA targeting huntingtin results in functionalimprovement, and therefore, represents a novel approach to providingeffective treatment of Huntington's disease.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of downregulating expression of a target gene in a neuralcell distal to the site of administration, the method comprisingcontacting an iRNA agent with the neural cell for a time sufficient toallow uptake of the iRNA agent into the cell, wherein (i) the iRNA agentcomprises a sense and an antisense strand that form an RNA duplex, and(ii) the sequence of the antisense strand of the iRNA agent comprises anucleotide sequence sufficiently complementary to a target sequence ofabout 18 to 25 nucleotides of an RNA expressed from the target gene. 2.A method of downregulating expression of a target gene in a neural cell,the method comprising contacting an iRNA agent with the neural cell fora time sufficient to allow uptake of the iRNA agent into the cell,wherein (i) the iRNA agent comprises a sense and an antisense strandthat form an RNA duplex, (ii) the iRNA agent comprises a lipophilicmoiety, and (iii) the sequence of the antisense strand of the iRNA agentcomprises a nucleotide sequence sufficiently complementary to a targetsequence of about 18 to 25 nucleotides of an RNA expressed from thetarget gene.
 3. The method of claim 1 or 2, wherein the cells arecontacted for a time sufficient to allow axonal transport of said iRNA.4. The method of claim 1, wherein the iRNA agent comprises a lipophilicmoiety.
 5. The method of claim 4, wherein the lipophilic moiety is acholesterol.
 6. The method of claim 4, wherein the lipophilic moiety isconjugated to the sense strand.
 7. The method of claim 4, wherein thelipophilic moiety is conjugated to the 3′ end of the sense strand.8.-12. (canceled)
 13. A method of treating a human comprisingidentifying a human having or at risk for developing a neurologicaldisorder, the method comprising administering to the human an iRNA agentthat targets a gene expressed in a neural cell distal to the site ofadministration, wherein the expression of the gene is associated withsymptoms of the neurological disorder, and wherein (i) the iRNA agentcomprises a sense and an antisense strand that form an RNA duplex, and(ii) the antisense strand of the iRNA agent comprises a nucleotidesequence sufficiently complementary to a target sequence of about 18 to25 nucleotides of an RNA expressed from the target gene.
 14. A method oftreating a human comprising identifying a human having or at risk fordeveloping a neurological disorder, and administering to the human aniRNA agent that targets a gene expressed in a neural cell, wherein theexpression of the gene is associated with symptoms of the neurologicaldisorder, and wherein (i) the iRNA agent comprises a sense and anantisense strand that form an RNA duplex, (ii) the iRNA agent comprisesa lipophilic moiety, and (iii) the antisense strand of the iRNA agentcomprises a nucleotide sequence sufficiently complementary to a targetsequence of about 18 to 25 nucleotides of an RNA expressed from thetarget gene.
 15. The method of claim 13, wherein the iRNA agentcomprises a lipophilic moiety.
 16. The method of claim 15, wherein thelipophilic moiety is a cholesterol.
 17. The method of claim 15, whereinthe lipophilic moiety is conjugated to the sense strand.
 18. The methodof claim 15, wherein the lipophilic moiety is conjugated to the 3′ endof the sense strand.
 19. The method of claim 15, wherein the iRNA agentfurther comprises a phosphorothioate or a 2′-OMe modification. 20.-33.(canceled)
 34. A method of reducing the amount of huntingtin (htt) RNAin a neural cell of a subject, comprising: contacting the neural cellwith an iRNA agent, wherein said neural cell is distal to the site ofaction and the iRNA agent comprises a sense and an antisense strand,wherein the sense and the antisense strands form an RNA duplex, whereinthe antisense strand comprises a nucleotide sequence that differs by nomore than four nucleotides from an antisense sequence listed in Table 1.35. The method of claim 34, wherein the iRNA agent further comprises alipophilic moiety.
 36. The method of claim 35, wherein the cells arecontacted for a time sufficient to allow axonal transport of said iRNA.37. The method of claim 36, wherein the iRNA agent further comprises aphosphorothioate or a 2′-OMe modification. 38.-42. (canceled)
 43. Anisolated iRNA agent comprising a sense and an antisense strand, whereinthe sense and the antisense strands form an RNA duplex, wherein theantisense strand comprises a nucleotide sequence that differs by no morethan four nucleotides from an antisense sequence listed in Table 1, andwherein the iRNA agent comprises a lipophilic moiety.
 44. The iRNA agentof claim 43, wherein the lipophilic moiety is a cholesterol molecule.45. The iRNA agent of claim 43, wherein the lipophilic moiety isattached to the sense strand.
 46. The iRNA agent of claim 43, whereinthe lipophilic moiety is attached to the 3′ end of the sense strand.47.-51. (canceled)
 52. A pharmaceutical composition, comprising: (i) aniRNA agent comprising a sense and an antisense strand, wherein the senseand the antisense strands form an RNA duplex, wherein the antisensestrand comprises a nucleotide sequence that differs by no more than fournucleotides from an antisense sequence listed in Table 1, and whereinthe iRNA agent comprises a lipophilic moiety; and (ii) apharmaceutically acceptable carrier. 53.-56. (canceled)
 57. A method ofevaluating an iRNA agent for enhanced uptake into neural cellscomprising: providing a candidate iRNA agent conjugated to a lipophilicagent, wherein the iRNA agent is in a solution that does not contain atransfection reagent, contacting the iRNA agent with a neural cell for atime sufficient for uptake into the neural cell and determining if theiRNA agent is taken up by the neural cell. 58.-66. (canceled)